Pacemaker ion channel proteins and uses thereof

ABSTRACT

The present invention provides an isolated nucleic acid molecule encoding a brain cyclic nucleotide gated ion channel (BCNG) protein. An isolated BCNG protein is also provided as is a composition comprising a BCNG encoding nucleic acid or protein or a portion thereof. The present invention also provides a method of identifying an ion channel subunit related protein encoding nucleic acid molecule in a sample. The invention further provides a method for evaluating the ability of a compound to modulate an ion channel associated neurological, cardia, or renal condition. The invention also provides a method for evaluating the ability of a compound to interact with a BCNG-related ion channel subunit protein. Additionally, the present invention provides a method for identifying a compound capable of modulating the activity of a BCNG-related protein. The present invention additionally provides a method of treating a cardiac, renal or neurological condition. Finally, the present invention provides an antibody which specifically reacts with BCNG protein.

Throughout this application, various publications are referenced byauthor and date. Full citations for these publications may be foundlisted alphabetically at the end of the specification immediatelypreceding the Sequence Listing and the claims. The disclosures of thesepublications in their entireties are hereby incorporated by referenceinto this application in order to more fully describe the state of theart.

BACKGROUND OF THE INVENTION

Introduction

Ion channels are a diverse group of proteins that regulate the flow ofions across cellular membranes. In the nervous system, ion channelactivity has evolved into a rapid and accurate system for intercellularcommunication. The electrical excitability characteristics of eachneuron is in part determined by the set of channels it expresses.However, cells are also able to regulate the activity of individualchannels in response to physiological or developmental events, and thereis growing evidence that ion channels can be the site of integration ofmultiple electrical and biochemical pathways.

In vivo, ion channels appear to be multimeric proteins that arecomprised of several distinct gene families, coding for channels withdistinct structural and functional properties. Within a gene family, thepotential for heterogeneity arising from the combinatorial assembly ofdifferent pore-forming and auxiliary subunits (Green, et al., 1995).Channel properties can be modulated by second messenger cascades and candirectly bind intracellular proteins such as kinases suggesting thatthis may be an important way to efficiently target the signaling cascadeto its effector molecule. The electrical characteristics of each neuronis, in part, determined by the set of ion channels that it expresses.However, cells are also able to regulate the activity of individualchannels in response to physiological or developmental events;pore-forming (α) subunits can interact with a variety of intracellularproteins, including auxiliary (β) subunits, cytoskeleton-associatedproteins and protein kinases (Green, et al., 1995). In addition toauxiliary (β) subunits, pore-forming subunits can interact with avariety of intracellular proteins and second messenger moleculesthemselves including G-proteins, cytoskeleton-associated proteins andprotein-kinases (Adelman, et al., 1995).

Several classes of ion channels bind directly, and are regulated by,second messenger molecules such as cyclic nucleotides (Zagotta, et al.,1996; Bruggemann, et al., 1993, and Hoshi, et al., 1995) or Ca⁺²(Adelman, et al., 1992; Kohler, et al., 1996). Channels with thisproperty may be key elements in the control of neuronal signaling, asthey directly couple biochemical cascades with electrical activity.Cyclic nucleotide-gated channels (CNG) play a distinct role both invisual and olfactory signal transduction; their recent identification inthe hippocampus and other regions of the brain, where cAMP and cGMP areknown to mediate different forms of synaptic plasticity (Krapivinisky,et al., 1995; Frey, et al., 1993; Boshakov, et al., 1997; and Arancio,et al., 1995), suggests that CNG-channels may also contribute to theregulation of excitability in central neurons (Kingston, et al., 1996and Bradley, et al., 1997).

The first structural gene for a K⁺ channel to be isolated was the geneencoded by the Shaker (Sh) locus in Drosophila melanogaster (Strong, etal., 1993; Papazian, et al., 1987). Its sequence is the prototype of alarge and still expanding family of related genes (Kamb, et al., 1987;Warmke, et al., 1994). The properties of a number of well characterizedK⁺ currents, that still await a molecular definition, predicts thatother members of this family are yet to be identified (Atkinson, et al.,1991).

Although the initial members of the K⁺ channel superfamily were clonedby chromosomal localization of alleles responsible for functionaldefects (Sh, eag and slo from Drosophila; (Papazian, et al., 1987; Kamb,et al., 1987; Warmke, et al., 1991; Atkinson, et al., 1991) or followingthe purification of a relatively abundant protein such as thecGMP-channel from bovine retina (Liman, et al., 1994), the most widelyused strategy for cloning new members of the K⁺ channel superfamily isby homology to these sequences. Unfortunately, this approach is not wellsuited for identifying more divergent sequences and potentially newbranches in the phylogenetic tree of the K⁺ channel superfamily.Expression cloning in Xenopus oocytes can circumvent this problem; thisimplies a pre-existing or readily detectable physiologicalcharacterization of the channel.

SUMMARY OF THE INVENTION

The present invention provides an isolated nucleic acid molecule havingsequence encoding a brain cyclic nucleotide gated (BCNG) ion channelprotein or a portion thereof. The present invention additionallyprovides an isolated BCNG protein. The present invention furtherprovides a composition comprising a nucleic acid, having sequenceencoding a BCNG protein or a portion thereof and a carrier. The presentinvention further additionally provides a composition comprising a BCNGprotein or portion thereof and a carrier.

In addition, the present invention provides a method of identifying anucleic acid encoding an ion channel subunit-related protein in a samplewhich comprises detecting in the sample a nucleic acid molecule encodinga BCNG-related protein, positive detection indicating the presence of anion channel subunit related protein encoding nucleic acid molecule,thereby identifying a nucleic acid encoding an ion channelsubunit-related protein in the sample.

The present invention also provides a method for evaluating the abilityof a compound to modulate an ion channel associated with a condition ina subject which comprises: (a) contacting a cell which expresses aBCNG-related protein in a cell culture with a compound; (b) determiningthe amount of BCNG-related protein expression in the cell culture; and(c) comparing the amount of BCNG-related protein expression determinedin step (b) with the amount determined in the absence of the compound soas to evaluate the ability of the compound to modulate the ion channelassociated with the condition in the subject.

The present invention also provides a method for evaluating the abilityof a compound to interact with a BCNG-related ion channel subunitprotein which comprises: (a) contacting a cell which expresses aBCNG-related protein in a cell culture with a compound under conditionspermissive to formation of a complex between the compound and theBCNG-related protein; (b) determining the amount of complex formedbetween the compound and the BCNG-related protein in the cell culture;and (c) comparing the amount of complex formed in step (b) with theamount formed in the absence of the compound so as to evaluate theability of the compound to interact with a BCNG-related ion channelsubunit protein.

Additionally, the present invention provides a method for identifyingwhether a compound is capable of modulating a dysfunction in an animalcomprising: (a) administering the compound to the animal underconditions permissive to formation of a complex between the compound andthe BCNG-related protein; (b) determining the amount of complex formedbetween the compound and the BCNG-related protein in the animal; and (c)comparing the amount of complex formed in step (b) with the amount ofcomplex formed in the animal in the absence of the compound so as toidentify whether the compound is capable of modulating the dysfunctionin the animal, a change in the amount of complex formed in the presenceof the compound indicating that the compound is capable of modulatingdysfunction in the animal.

Further, the present invention provides a method of identifying acompound which modulates the activity of BCNG-related protein whichcomprises: (a) introducing the nucleic acid of claim 1 into anexpression system and causing the expression system to express thenucleic acid under conditions whereby an ion channel subunit protein isproduced; (b) contacting the channel subunit protein with the compound;(c) determining the activity of the ion channel subunit protein; and (d)comparing the activity determined in step (c) with the activitydetermined in the absence of the compound, an increase or decrease inactivity in the presence of the compound indicating identification of acompound which modulates the activity of BCNG-related protein.

The present invention also provides a pharmaceutical composition whichcomprises a compound capable of modulating BCNG-related protein activityand a pharmaceutically acceptable carrier. According to an embodiment ofthis invention, the carrier is a diluent, an aerosol, a topical carrier,an aqueous solution, a nonaqueous solution or a solid carrier.

Finally, the present invention provides an antibody that specificallybinds to BCNG protein.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1D. Primary structure of BCNG-1. (FIG. 1A) Deduced amino acidsequence encoded by the BCNG-1 cDNA. The seven hydrophobic domains,homologous to the six transmembrane domains (S1-S6) and the pore (P) ofK⁺ channels, are indicated (______). The putative cyclic-nucleotidebinding site (CNBs) is marked by an ( - - - ), C-terminal prolines ( . .. ) the consensus N-glycosylation site with presumptive extracellularlocalization (*) are also marked. (FIG. 1B) Kyte and Doolittlehydropathy plot of the predicted amino acid sequence of BCNG-1. Theprofile was generated by the Kyte and Doolittle method with a windowsize of 7 amino acids. The numbers on the top line indicate the positionin the BCNG-1 sequence. Hydrophobic regions corresponding to S1 throughS6 and the P region lie below the zero line while the N-glycosylationsite (*) is in a hydrophilic region between S5 and P. Numbering (topline) indicate position in the BCNG-1 sequence. Profile generated with awindow size of 7 residues. (FIG. 1C) Multiple alignment of the putativeP region of BCNG-1 with the P regions of Drosophila Eag (DEAG), mouseEag (MEAG), human Erg (HERG), α-subunit of bovine retinal CNG-channel(BRET-1), and β-subunit of human retinal CNG-channel (HRET-2).Arrowheads mark the residues 344 and 352 (see Example 1). (FIG. 1D)Alignment of the (CNBs) of BCNG-1 with the corresponding site in the ratolfactory CNG-channel (ROLF-1), bovine cGMP-dependent protein kinase(PKG), bovine cAMP-dependent protein kinase (PKA), and cataboliteactivator protein of E. coli (CAP). Continuous lines mark α-helical (a)and β-strand (β) elements of the secondary structure elements of CAP,while asterisks indicate specific amino acids that appear to lie closeto the cAMP molecule in the CAP crystal structure.

FIGS. 2A-2D. BCNG-1 is a 132 kDa glycosylated protein. (FIG. 2A) Westernblot analysis of BCNG-1 protein in a mouse brain extract. Ten μg of atotal brain SDS-extract was loaded per strip then probed with αq1 (1) orαq2 (3) antiserum or, strip 2 with αq1 (2) or αq2 (4) antiserumpreadsorbed with the GST-d5 fusion protein. The arrow marks the positionof the specific signal, of corresponding to the native BCNG-1 protein.(FIG. 2B) Western blot using the αq1 antiserum against: total brainextract (1), total brain extract pretreated with N-glycosidase F (2) andin vitro translated BCNG-1 protein (3). Positions of molecular weightstandards are shown on the left. Also shown, Western blot containing 10μg of proteins from each of the indicated brain tissues which was testedwith antisera against mBCNG-1 and showing widespread expression of themBCNG-1 protein in mouse brain. (FIG. 2C) indicates reactivity with αq1.(FIG. 2D) indicates reactivity with αq2.

FIG. 3. Northern blot analysis of BCNG-1 expression in different mousetissues. Two μg of poly(A)⁺ RNA from each of each of the followingtissues was used: heart (H), brain (B), spleen (S), lung (Lu), liver(Li), skeletal muscle (M), kidney (K) and testis (T) were loaded. Thefilter was probed with a DNA fragment encoding amino acids 6-131 of theBCNG-1 sequence. A probe corresponding to amino acids 594-720 recognizedthe same bands, confirming that the cDNA fragments isolated from theλgt10 and pJG4-5 libraries are from a contiguous mRNA sequence.Positions of molecular weight standards are shown on the left.

FIG. 4. In situ hybridization analysis of BCNG-1 expression in thebrain. Parasagittal section of a mouse brain probed with an antisenseoligonucleotide directed to the mRNA region corresponding to amino acids648-657 of the BCNG-1 sequence. Abbreviations: nCtx, neocortex; Hp,hippocampus; Crb, cerebellum; BrSt, brainstem.

FIGS. 5A-5F: Immunohistochemical analysis of BCNG-1 expression in thebrain. Parasagittal sections of a mouse brain were stained with αq1 andαq2 antisera. The patterns of BCNG-1 expression detected with the twodifferent antisera were identical, and in both cases the staining wasentirely abolished by preadsorbing the sera with the GST-d5 fusionprotein. BCNG-1 immunoreactivity in the cerebral cortex. (FIG. 5C), and(FIG. 5D): BCNG-1 immunoreactivity in the hippocampus. In (FIG. 5C), thearrow shows the position of the hippocampal fissure; areas CA₁, CA₃ anddentate gyrus (DG) are labeled. and; (FIG. 5D) shows a detail of thestratum pyramidale of area CA₃. (FIG. 5E) and (FIG. 5F): BCNG-1immunoreactivity in the cerebellum (FIG. 5A, FIG. 5C, FIG. 5E: 60×; FIG.5B, FIG. 5D, FIG. 5F: 100×) magnification).

FIGS. 6A-6B. Southern blot analysis of mouse genomic DNA. 4 μg of mousegenomic DNA were loaded onto each lane following digested with Eco RI(1), Hind III (2), Bam HI (3), Pst I (4) or Bgl II (5). The filter wasprobed with a DNA fragment encoding amino acids 269-462 of the BCNG-1sequence at high (FIG. 6A) and (FIG. 6B) low stringency. Positions ofmolecular weight standards are shown on the left.

FIGS. 7A-7B. The predicted structure of mBCNG-1 with six transmembranesequences (S1-S6), the pore region (P), the cyclic nucleotide bindingsite and long C-terminal tail including a poly-glutamine stretch (Q).The proteins predicted to be encoded by the partial fragments of theother BCNG genes that have been shown to be aligned to mBCNG-1. Doubleheaded arrow lines above the sequences indicate if the relative fragmentwas obtained from a cDNA library (λgt10 or pJG4-5), RT-PCR reaction, orEST database. Double headed dashed arrow lines underneath the sequencesindicate the position of the different probes that were used in thisstudy. On the right of the sequences, the percent of amino acidsimilarity to the mBCNG-1 protein is reported (mBNCG-2 where indicated).The alignments were performed by comparing only the core region of theproteins, including transmembrane domains S1-S6, corresponding to aminoacids 111-419 (numbering according to mBCNG-1). The mBCNG-4 sequence wasnot included in this alignment (ND, not determined). However, limitedalignment of the available cyclic nucleotide binding domain sequence(amino acids 529-592, number according to mBCNG-1) shows a 79%similarity to mBCNG-1. Hashed box near mBCNG-4 sequence indicatesposition of probable intron found in the M28-EST clone.

FIGS. 8A-8B. Alignment of the predicted amino acid sequences for fourmouse genes (mBCNG-1, 2, 3 and 4) and two human genes (hBCNG-1 and 2).The proposed structural features of the protein (putative transmembraneregions, the pore region and the cyclic nucleotide binding site) areindicated. (-) indicates residues identical to mBCNG-1; divergentresidues are otherwise reported. (x) indicates residues not determined.(.) indicates a gap (or deletion) in the aligned sequence. (*) at theend of the sequence indicates a stop codon. (*) above position 327 marksN-glycosylation site of mBCNG-1.

FIGS. 9A-9D. Northern blot analysis of mouse BCNG genes. A MultipleTissue Northern blot, containing 2 mg of polyA⁺ RNA from each of thefollowing mouse tissues: heart (He), brain (Br), spleen (Sp), lung (Lu)liver (Li), skeletal muscle (Mu), kidney (Ki) and testis (Te), washybridized to DNA fragments corresponding to the indicated BCNG genes.Molecular size markers are indicated on the left.

FIGS. 10A-10B. Northern blot analysis of human BCNG genes. A MultipleTissue Northern blot, containing 2 mg of polyA⁺ RNA from each of thefollowing human tissues: heart (He), brain (Br), placenta (Pl), lung(Lu) liver (Li), skeletal muscle (Mu), kidney (Ki) and pancreas (Pa),was hybridized to DNA fragments corresponding to the indicated BCNGgenes. The same fragments were used to probe a human Brain MultipleTissue blot, containing 2 mg of polyA⁺ RNA from each of the followingtissues: amygdala (Am), caudate nucleus (Cn), corpus callosum (CC),hippocampus (Hi), total brain (Br), substantia nigra (SN), subthalamicnucleus (Sn) and thalamus (Th). Molecular size markers are indicated onthe left.

FIG. 11. Schematic representation of the general architecture of theBCNG channel proteins based on homology to the voltage-gated K⁺ channelsand the cyclic nucleotide-gated channels.

FIG. 12. Alignment of the S4 voltage sensing regions of the prototypicalvoltage—gated K⁺ channel shaker and cyclic nucleotide-gated channelbRET1 with the S4 sequence of mBCNG1. Boxed residues are positivelycharged amino acids present in one or more of the S4 sequences. Thestars indicate the position of amino acids with negatively chargedacidic side chains that are present in the bRET1 sequence.

FIG. 13A-13B. (FIG. 13A) Sequence alignment of functional cyclicnucleotide binding sites from catabolite activating protein (CAP, Aibaet al., 1982; Cossart & Gicquel, 1982), A and B sites of recombinantbovine R1α (PKAa and PKAb, Titani et al., 1984), bovine retinal channelα subunit (bRET1, Kaupp et al., 1989) and the catfish olfactory αsubunit (fOLF1, Goulding et al., 1992) along with the putative cyclicnucleotide binding sites of drosophila Ether-a-gogo (dEAG, Warmke etal., 1991), Arabidopsis Thaliana K transport protein (KAT1, Anderson etal., 1992) and the mouse BCNG-1 channel (mBCNG-1, described herein). Thesix residues that are totally conserved across all of the binding siteswhose functional competence has been unequivocally confirmed are markedby asterisks. The conserved arginine that forms an ionic bond with thecyclic nucleotide is indicated by an arrow labeled R559. The residue inthe third (C) α-helix that has been shown to influence coupling ofactivation to cAMP versus cGMP binding is indicated by an arrow labeledD604 (the cGMP selective substitution in bRET1).

(FIG. 13B) Schematic representation of the cyclic nucleotide bindingsite of bRET1 showing the critical interactions between the binding siteand the cyclic nucleotide. This model of the binding pocket is based onthe crystal structure of CAP and bovine R1α. The cGMP is shown bound inan extended—or anti—form with the cyclized phosphate making an ionicbond with Arginine559 (bRET1 numbering) and the purine ring formingfavorable contacts with D604 in this cGMP selective channel.

FIG. 14. Alignment of the P loop pore forming regions of theprototypical voltage-gated K⁺ channel shaker and cyclic nucleotide-gatedchannel bRET1 with the S4 sequence of mBCNG1. The aligned channels aremouse BCNG-1 (BCNG-1), Shaker (SHAK, Papazian et al., 1987; Kamb et al.,1988), SHAW, Wei, et al., 1990) calcium activated K channel (MSLO,Pallanck and Ganetzky, 1994) the plant inward rectifier (AKT Sentenac etal., 1992), drosophila and mouse ether-a-gogo's (DEAG, Warmke et al.,1991; MEAG, Warmke and Ganetzky, 1994) Human ether-a-gogo related gene(HERG, Warmke and Ganetzky, 1994), bovine retinal α-subunit (bRET1,Kaupp et al., 1989) and human retinal β subunit (HRET-2, Chen et al.,1993). The arrows mark positions where the BCNG channels show pronouncedand potentially important changes in sequence from the normal motif seenin K selective channels.

FIG. 15A-15B. Schematic representation of repetitive firing of apacemaker neuron and its involvement in the generation and regulation ofrhythmic firing patterns. (FIG. 15A) Shows that Ih activation uponhyperpolarization following an action potential. As this is anon-selective cationic current, it carries an inward current at thesepotentials which leads to the depolarization of the cell back towardsthe threshold for firing of the next action potential. (FIG. 15B) Showsthe action of sympathetic and vagal stimulation on the activity ofcardiocytes from the sinoatrial node—the pacemaker area of the heart.Norepinephrine (NE) leads to a shift in the activation of Ih towardsmore depolarized potentials which accelerates the return to the actionpotential firing threshold, and hence, leads to an acceleration of thefiring rate. In contrast, acetylcholine (ACh) shifts the activation ofIh to more hyperpolarized potentials. Thus, the current will turn onlater during the repolarization phase delaying the return tothreshold—the firing rate of the cell will thus be slowed. These changesin the activation properties of Ih are thought to be due to changes inthe concentration of cAMP with ACh lowering the concentration and NEincreasing the concentration which has been shown to alter theactivation properties of Ih. (“Principles of Neural Science” by Kandel,Schwartz and Jessell 1991).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an isolated nucleic acid molecule havingsequence encoding a BCNG ion channel protein or a portion thereof. Inone embodiment of the present invention, the nucleic acid molecule ismBCNG-1, mBCNG-2, mBCNG-3, mBCNG-4, hBCNG-1 or hBCNG-2. In an embodimentof the present invention, the nucleic acid molecule is DNA. In anotherembodiment of the invention, the nucleic acid molecule is RNA. In yetanother embodiment of the present invention, the DNA is cDNA. In oneembodiment of this invention, the cDNA has substantially the same codingsequence as the coding sequence shown in SEQ. ID. No.: 1, SEQ. ID. No.:3, SEQ. ID. No.: 5, SEQ. ID. No.: 7, SEQ. ID. No.: 9, or SEQ. ID.No.:11. In still another embodiment the present invention is a vectorcomprising the nucleic acid. In one embodiment of this invention, thevector is a virus. In another embodiment of this invention, the vectoris a plasmid. Another embodiment of this invention is a host vectorsystem for the production of a mammalian BCNG molecule which comprisesthe vector in a suitable host. In an embodiment of this invention, thesuitable host is a bacterial cell, a eukaryotic cell, a mammalian cellor an insect cell. Another embodiment of the present invention is anucleic acid molecule capable of specifically hybridizing with thenucleic acid molecule encoding BCNG-related protein. Still anotherembodiment of the present invention is an isolated BCNG protein. In anembodiment of this invention, the BCNG protein is mBCNG-1, mBCNG-2,mBCNG-3, mBCNG-4, hBCNG-1 or hBCNG-2. In another embodiment of thisinvention, the protein has substantially the same amino acid sequence asthe amino acid sequence shown in SEQUENCE ID NO.: 2, SEQ. ID. No.: 4,SEQ. ID. No.: 6, SEQ. ID. No.: 8, SEQ. ID. No.: 10, or SEQ. ID. No.: 12.Still another embodiment of the present invention is a compositioncomprising a nucleic acid, having sequence encoding a BCNG protein or aportion thereof and a carrier. In an embodiment of this invention, thenucleic acid comprises substantially the same coding sequence as thecoding sequence shown in SEQ. ID. No.: 1, SEQ. ID. No.: 3, SEQ. ID. No.:5, SEQ. ID. No.: 7, SEQ. ID. No.: 9, SEQ. ID. No.:11 or portion thereof.Yet another embodiment of the present invention is a compositioncomprising a BCNG protein or portion thereof and a carrier. In oneembodiment of this invention, the BCNG protein comprises substantiallythe same amino acid sequence as the amino acid sequence shown in SEQ.ID. No.: 2, SEQ. ID. No.: 4, SEQ. ID. No.: 6, SEQ. ID. No.: 8, SEQ. ID.No.: 10, SEQ. ID. No.:12 or a portion thereof.

In addition, the present invention provides a method of identifying anucleic acid encoding an ion channel subunit-related protein in a samplewhich comprises detecting in the sample a nucleic acid molecule encodinga BCNG-related protein, positive detection indicating the presence of anion channel subunit related protein encoding nucleic acid molecule,thereby identifying a nucleic acid encoding an ion channelsubunit-related protein in the sample.

In an embodiment of the present invention, the nucleic acid moleculeencoding the ion channel subunit related protein is detected by sizefractionation. In an embodiment of this invention, the sizefractionation is effected by a polyacrylamide or an agarose gel. In anembodiment of this invention, the detection of the nucleic acid moleculeencoding the BCNG-related protein comprises contacting the sample with asecond nucleic acid molecule capable of hybridizing with the nucleicacid molecule encoding the BCNG-related protein, wherein the secondnucleic acid molecule is labeled with a detectable marker underconditions permitting the second nucleic acid molecule to hybridize withthe nucleic acid molecule encoding the BCNG-related protein, therebyidentifying the nucleic acid molecule in the sample. In one embodimentof this invention, the second nucleic acid molecule is capable ofhybridizing to mBCNG-1, mBCNG-2, mBCNG-3, mBCNG-4, hBCNG-1 or hBCNG-2.In one embodiment of the invention, the detectable marker is aradiolabeled molecule, a fluorescent molecule, an enzyme, a ligand, or amagnetic bead. In an embodiment of this invention, the detection of thenucleic acid molecule encoding the BCNG-related protein comprisesamplifying the nucleic acid molecule encoding the BCNG-related protein,thereby identifying the nucleic acid molecule encoding the BCNG-relatedprotein in the sample. In one embodiment of the invention, theamplification of the nucleic acid molecule encoding the BCNG-relatedprotein comprises contacting the nucleic acid molecule from the samplewith at least one primer capable of hybridizing to mBCNG-1, mBCNG-2,mBCNG-3, mBCNG-4, hBCNG-1 or hBCNG-2 under conditions suitable forpolymerase chain reaction. In an embodiment of this invention, theamplified nucleic acid molecule encoding the BCNG-related protein isdetected by size fractionation. In an embodiment of this invention, thesize fractionation is via a polyacrylamide gel or an agarose gel.

The present invention also provides a method for evaluating the abilityof a compound to modulate an ion channel associated with a condition ina subject which comprises: (a) contacting a cell which expresses aBCNG-related protein in a cell culture with a compound; (b) determiningthe amount of BCNG-related protein expression in the cell culture; and(c) comparing the amount of BCNG-related protein expression determinedin step (b) with the amount determined in the absence of the compound soas to evaluate the ability of the compound to modulate the ion channelassociated with the condition in the subject.

In an embodiment of this invention, the condition is a neurologicalcondition, cardiovascular condition, renal condition, pulmonarycondition, or hepatic condition. In one embodiment of this invention,the condition is epilepsy, Alzheimer's Disease, Parkinson's Disease,long QT syndrome, sick sinus syndrome, age-related memory loss, cysticfibrosis, sudden death syndrome or a pacemaker rhythm dysfunction.According to an embodiment of this invention, the BCNG-related proteincomprises mBCNG-1, mBCNG-2, MBCNG-3, mBCNG-4, hBCNG-1 or hBCNG-2 or aportion thereof. In an embodiment of this invention, the cell is acardiac cell, a kidney cell, a hepatic cell, an airway epithelial cell,a muscle cell, a neuronal cell, a glial cell, a microglial cell, anendothelial cell, a mononuclear cell, a tumor cell, a mammalian cell, aninsect cell, or a Xenopus oocyte. According to an embodiment of thisinvention, the compound is a peptide, a peptidomimetic, a nucleic acid,a polymer, or a small molecule. In one embodiment of this invention, thecompound is bound to a solid support.

The present invention also provides a method for evaluating the abilityof a compound to interact with a BCNG-related ion channel subunitprotein which comprises: (a) contacting a cell which expresses aBCNG-related protein in a cell culture with a compound under conditionspermissive to formation of a complex between the compound and theBCNG-related protein; (b) determining the amount of complex formedbetween the compound and the BCNG-related protein in the cell culture;and (c) comparing the amount of complex formed in step (b) with theamount formed in the absence of the compound so as to evaluate theability of the compound to interact with a BCNG-related ion channelsubunit protein. According to one embodiment of this invention, theBCNG-related protein comprises mBCNG-1, mBCNG-2, mBCNG-3, mBCNG-4,hBCNG-1 or hBCNG-2 or a portion thereof. In an embodiment of the presentinvention, the cell is a cardiac cell, a kidney cell, a hepatic cell, anairway epithelial cell, a muscle cell, a neuronal cell, a glial cell, amicroglial cell, an endothelial cell, a mononuclear cell, a tumor cell,a mammalian cell, an insect cell, or a Xenopus oocyte. Further, in anembodiment of this invention, the compound is a peptide, apeptidomimetic, a nucleic acid, a polymer, or a small molecule. In stilla further embodiment of the present invention, the compound is bound toa solid support.

Additionally, the present invention provides a method for identifyingwhether a compound is capable of modulating a dysfunction in an animalcomprising: (a) administering the compound to the animal underconditions permissive to formation of a complex between the compound andthe BCNG-related protein; (b) determining the amount of complex formedbetween the compound and the BCNG-related protein in the animal; and (c)comparing the amount of complex formed in step (b) with the amount ofcomplex formed in the animal in the absence of the compound so as toidentify whether the compound is capable of modulating the dysfunctionin the animal, a change in the amount of complex formed in the presenceof the compound indicating that the compound is capable of modulatingdysfunction in the animal. In one embodiment of this invention is a thecondition is a neurological condition, cardiovascular condition, renalcondition, pulmonary condition, or hepatic condition. In yet anotherembodiment, the condition is epilepsy, Alzheimer's Disease, Parkinson'sDisease, long QT syndrome, sick sinus syndrome, age-related memory loss,cystic fibrosis, sudden death syndrome or a pacemaker rhythmdysfunction.

Further, the present invention provides a method of identifying acompound which modulates the activity of BCNG-related protein whichcomprises: (a) introducing the nucleic acid of claim 1 into anexpression system and causing the expression system to express thenucleic acid under conditions whereby an ion channel subunit protein isproduced; (b) contacting the channel subunit protein with the compound;(c) determining the activity of the ion channel subunit protein; and (d)comparing the activity determined in step (c) with the activitydetermined in the absence of the compound, an increase or decrease inactivity in the presence of the compound indicating identification of acompound which modulates the activity of BCNG-related protein.

In one embodiment of this invention, the active determination step (c)comprises measuring the channel electrical current or intracellularcalcium level in the presence of the compound. In another embodiment,the detection is by a florescence-activated cell sorter (FACS) or by avideo imaging system. In another embodiment of this invention, thedetection is in a high throughput screening system. In anotherembodiment of this invention, the expression system comprises a culturedhost cell. In another embodiment, the host cell is a cardiac cell, akidney cell, a hepatic cell, an airway epithelial cell, a muscle cell, aneuronal cell, a glial cell, a microglial cell, an endothelial cell, amononuclear cell, a tumor cell, a mammalian cell, an insect cell, or aXenopus oocyte.

The present invention also provides a pharmaceutical composition whichcomprises a compound capable of modulating BCNG-related protein activityand a pharmaceutically acceptable carrier. According to an embodiment ofthis invention, the carrier is a diluent, an aerosol, a topical carrier,an aqueous solution, a nonaqueous solution or a solid carrier.

One embodiment of this invention is a method for treating a condition ina subject which comprises administering to the subject an amount of thepharmaceutical composition effective to treat the condition in thesubject. In an embodiment of this invention, the condition is aneurological condition, renal condition, pulmonary condition, hepaticcondition, or cardiovascular condition. In an embodiment of thisinvention, the condition is epilepsy, Alzheimer's Disease, Parkinson'sDisease, long QT syndrome, sick sinus syndrome, age-related memory loss,cystic fibrosis, sudden death syndrome or a pacemaker rhythmdysfunction. In still another embodiment of this invention, the subjectis a human.

The present invention further provides a method of treating a conditionin a subject which comprises administering to the subject an effectiveamount of a compound which modulates the activity of BCNG-relatedprotein, so as to treat the condition. In an embodiment of thisinvention, the condition is a neurological condition, cardiovascularcondition, renal condition, pulmonary condition, or hepatic condition.In another embodiment of this invention, the condition is epilepsy,Alzheimer's Disease, Parkinson's Disease, long QT syndrome, sick sinussyndrome, age-related memory loss, cystic fibrosis, sudden deathsyndrome or a pacemaker rhythm dysfunction.

Finally, the present invention provides an antibody that specificallybinds to BCNG protein. One embodiment of this invention is a cell lineproducing the antibody specific for BCNG-related protein. Anotherembodiment of the present invention is a method of identifying theprotein of claim 11 in a sample comprising: a) contacting the samplewith the antibody of claim 56 under conditions permissive to theformation of a complex between the antibody and the protein; b)determining the amount of complex formed; and c) comparing the amount ofcomplex formed in step (b) with the amount of complex formed in theabsence of the antibody, the presence of an increased amount of complexformed in the presence of the antibody indicating identification of theprotein in the sample.

The degree of hybridization depends on the degree of complementarity,the length of the nucleic acid molecules being hybridized, and thestringency of the conditions in a reaction mixture. Stringencyconditions are affected by a variety of factors including, but notlimited to temperature, salt concentration, concentration of the nucleicacids, length of the nucleic acids, sequence of the nucleic acids andviscosity of the reaction mixture. More stringent conditions requiregreater complementarity between the nucleic acids in order to achieveeffective hybridization.

A preferred method of hybridization is blot hybridization. See Sambrooket al. 1989 Molecular Cloning: A Laboratory Manual 2nd Ed. foradditional details regarding blot hybridization.

The second nucleic acid molecule specifically hybridizes to a particularsequence which is bound nonspecifically to the solid matrix. The secondnucleic acid molecule can be DNA or RNA and can be labeled with adetectable marker. Such labeling techniques methods include, but are notlimited to, radio-labeling, digoxygenin-labeling, and biotin-labeling.

A well-known method of labeling DNA is ³²P using DNA polymerase, Klenowenzyme or polynucleotide kinase. In addition, there are knownnon-radioactive techniques for signal amplification including methodsfor attaching chemical moieties to pyrimidine and purine rings (Dale, R.N. K. et al., 1973 Proc. Natl. Acad. Sci. USA 70:2238-42), methods whichallow detection by chemiluminescence (Barton, S. K. et al., 1992 J. Am.Chem. Soc. 114:8736-40) and methods utilizing biotinylated nucleic acidprobes (Johnson, T. K. et al., 1983 Anal. Biochem. 133:125-131;Erickson, P. F. et al., 1982 J. Immunol. Methods 51:241-49; Matthaei, F.S. et al., 1986 Anal. Biochem. 157-123-28) and methods which allowdetection by fluorescence using commercially available products.Non-radioactive labelling kits are also commercially available.

Nucleic acid amplification is described in Mullis, U.S. Pat. No.4,683,202, which is incorporated herein by reference.

In a polymerase chain reaction (PCR), an amplification reaction uses atemplate nucleic acid contained in a sample can use one or more primersequences and inducing agents.

Suitable enzymes to effect amplification, specifically extensioninclude, for example, E. coli DNA polymerase I, thermostable Taq DNApolymerase, Klenow fragment of E. coli DNA polymerase I, T4 DNApolymerase, other available DNA polymerases, reverse transcriptase andother enzymes which will facilitate covalent linkage of the nucleotidesto polynucleotides which are form amplification products. Theoligonucleotide primers can be synthesized by automated instruments soldby a variety of manufacturers or can be commercially prepared.

Solid matrices are available to the skilled artisan. A solid matrix mayinclude polystyrene, polyethylene, polypropylene, polycarbonate, or anysolid plastic material in the shape of test tubes, beads,microparticles, dipsticks, plates or the like. Additionally matricesinclude, but are not limited to membranes, 96-well microtiter plates,test tubes and Eppendorf tubes. Solid phases also include glass beads,glass test tubes and any other appropriate shape made of glass. Afunctionalized solid phase such as plastic or glass which has beenmodified so that the surface carries carboxyl, amino, hydrazide, oraldehyde groups can also be used. In general such matrices comprise anysurface wherein a ligand-binding agent can be attached or a surfacewhich itself provides a ligand attachment site.

In the practice of any of the methods of the invention or in thepreparation of any of the pharmaceutical compositions of the presentinvention a “therapeutically effective amount” is an amount which iscapable of modulating the activity or function of a BCNG-relatedprotein. Accordingly, the effective amount will vary with the subjectbeing treated, as well as the condition to be treated. The methods ofadministration may include, but are not limited to, administrationcutaneously, subcutaneously, intravenously, parenterally, orally,topically, or by aerosol.

As used herein, the term “suitable pharmaceutically acceptable carrier”encompasses any of the standard pharmaceutically accepted carriers, suchas phosphate buffered saline solution, water, emulsions such as anoil/water emulsion or a triglyceride emulsion, various types of wettingagents, tablets, coated tablets and capsules. An example of anacceptable triglyceride emulsion useful in intravenous andintraperitoneal administration of the compounds is the triglycerideemulsion commercially known as Intralipid®.

Typically such carriers contain excipients such as starch, milk, sugar,certain types of clay, gelatin, stearic acid, talc, vegetable fats oroils, gums, glycols, or other known excipients. Such carriers may alsoinclude flavor and color additives or other ingredients.

This invention also provides for pharmaceutical compositions capable ofinhibiting neurotoxicity together with suitable diluents, preservatives,solubilizers, emulsifiers, adjuvants and/or carriers. Such compositionsare liquids or lyophilized or otherwise dried formulations and includediluents of various buffer content (e.g., Tris-HCl., acetate,phosphate), pH and ionic strength, additives such as albumin or gelatinto prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80,Pluronic F68, bile acid salts), solubilizing agents (e.g., glycerol,polyethylene glycerol), anti-oxidants (e.g., ascorbic acid, sodiummetabisulfite), preservatives (e.g., Thimerosal, benzyl alcohol,parabens), bulking substances or tonicity modifiers (e.g., lactose,mannitol), covalent attachment of polymers such as polyethylene glycolto the compound, complexation with metal ions, or incorporation of thecompound into or onto particulate preparations of polymeric compoundssuch as polylactic acid, polglycolic acid, hydrogels, etc, or ontoliposomes, micro emulsions, micelles, unilamellar or multi lamellarvesicles, erythrocyte ghosts, or spheroplasts. Such compositions willinfluence the physical state, solubility, stability, rate of in vivorelease, and rate of in vivo clearance of the compound or composition.

Controlled or sustained release compositions include formulation inlipophilic depots (e.g., fatty acids, waxes, oils). Also comprehended bythe invention are particulate compositions coated with polymers (e.g.,poloxamers or poloxamines) and the compound coupled to antibodiesdirected against tissue-specific receptors, ligands or antigens orcoupled to ligands of tissue-specific receptors. Other embodiments ofthe compositions of the invention incorporate particulate formsprotective coatings, protease inhibitors or permeation enhancers forvarious routes of administration, including parenteral, pulmonary, nasaland oral.

When administered, compounds are often cleared rapidly from thecirculation and may therefore elicit relatively short-livedpharmacological activity. Consequently, frequent injections ofrelatively large doses of bioactive compounds may by required to sustaintherapeutic efficacy. Compounds modified by the covalent attachment ofwater-soluble polymers such as polyethylene glycol, copolymers ofpolyethylene glycol and polypropylene glycol, carboxymethyl cellulose,dextran, polyvinyl alcohol, polyvinylpyrrolidone or polyproline areknown to exhibit substantially longer half-lives in blood followingintravenous injection than do the corresponding unmodified compounds(Abuchowski et al., 1981; Newmark et al., 1982; and Katre et al., 1987).Such modifications may also increase the compound's solubility inaqueous solution, eliminate aggregation, enhance the physical andchemical stability of the compound, and greatly reduce theimmunogenicity and reactivity of the compound. As a result, the desiredin vivo biological activity may be achieved by the administration ofsuch polymer-compound adducts less frequently or in lower doses thanwith the unmodified compound.

Attachment of polyethylene glycol (PEG) to compounds is particularlyuseful because PEG has very low toxicity in mammals (Carpenter et al.,1971). For example, a PEG adduct of adenosine deaminase was approved inthe United States for use in humans for the treatment of severe combinedimmunodeficiency syndrome. A second advantage afforded by theconjugation of PEG is that of effectively reducing the immunogenicityand antigenicity of heterologous compounds. For example, a PEG adduct ofa human protein might be useful for the treatment of disease in othermammalian species without the risk of triggering a severe immuneresponse. The carrier includes a microencapsulation device so as toreduce or prevent an host immune response against the compound oragainst cells which may produce the compound. The compound of thepresent invention may also be delivered microencapsulated in a membrane,such as a liposome.

Polymers such as PEG may be conveniently attached to one or morereactive amino acid residues in a protein such as the alpha-amino groupof the amino terminal amino acid, the epsilon amino groups of lysineside chains, the sulfhydryl groups of cysteine side chains, the carboxylgroups of aspartyl and glutamyl side chains, the alpha-carboxyl group ofthe carboxy-terminal amino acid, tyrosine side chains, or to activatedderivatives of glycosyl chains attached to certain asparagine, serine orthreonine residues.

Numerous activated forms of PEG suitable for direct reaction withproteins have been described. Useful PEG reagents for reaction withprotein amino groups include active esters of carboxylic acid orcarbonate derivatives, particularly those in which the leaving groupsare N-hydroxysuccinimide, p-nitrophenol, imidazole or1-hydroxy-2-nitrobenzene-4-sulfonate. PEG derivatives containingmaleimido or haloacetyl groups are useful reagents for the modificationof protein free sulfhydryl groups. Likewise, PEG reagents containingamino hydrazine or hydrazide groups are useful for reaction withaldehydes generated by periodate oxidation of carbohydrate groups inproteins.

This invention is illustrated in the Experimental Details section whichfollows. These sections are set forth to aid in an understanding of theinvention but are not intended to, and should not be construed to, limitin any way the invention as set forth in the claims which followthereafter.

EXPERIMENTAL DETAILS Example 1 Interactive Cloning with the SH3 Domainof N-Src Identifies a New Brain-Specific Ion Channel Protein, withHomology to Cyclic Nucleotide-Gated Channels

By screening for molecules that interact with the neuronal form of Srctyrosine kinase a novel cDNA was isolated that appears to represent anew class of ion channels. The encoded polypeptide, mBCNG-1, isdistantly related to proteins in the family of the cyclicnucleotide-gated channels and the voltage-gated channels, Eag and H-erg.mBCNG-1 is expressed exclusively in the brain as a glycosylated proteinof approximately 132 kD. Immunohistochemical analysis indicates thatmBCNG-1 is preferentially expressed in specific subsets of neurons inthe neocortex, hippocampus and cerebellum, in particular pyramidalneurons and basket cells. Within individual neurons, the BCNG-1 proteinis localized to either the dendrites or the axon terminals depending onthe cell type.

Southern blot analysis shows that several other BCNG-related sequencesare present in the mouse genome, indicating the emergence of an entirelynew subfamily of ion channel coding genes. These findings suggest theexistence of a novel class of ion channel, which is potentially able tomodulate membrane excitability in the brain and which may respond toregulation by cyclic nucleotides.

Defining signal transduction pathways that contribute to the control ofsynaptic strength in the brain is an important and long-sought goal. Inan effort to identify the biochemical targets of Src-family tyrosinekinases in the central nervous system, the yeast two-hybrid system wasused to clone proteins that could interact with the SH3 domain of theneural specific form of Src kinase (Brugge, et al., 1985; Martinez, etal., 1987). As a result of this screening a new protein, BCNG-1 (BrainCyclic Nucleotide Gated-1) was identified and isolated.

BCNG-1 has been identified and characterized as an ion channel proteinand exhibits sequence homology to voltage-gated potassium channels, CNGchannels, and plant inward rectifers. Southern blot analysis suggeststhat this is the first member of a new family of proteins. BCNG-1 isexpressed exclusively in the brain and is preferentially localized tothe processes of subsets of neurons in the neocortex, cerebellar cortexand hippocampus. The specific localization pattern of BCNG-1 and thepotential for a direct interaction with cyclic nucleotides suggest thatit may represent a new brain-specific ion channel protein that is animportant component in the expression of intercellular and intracellularsignaling.

Results

Isolation of BCNG-1. BCNG-1, a novel cDNA with homology to CNG-relatedand Eag-related ion channels was initially isolated and identified byinteractive cloning with the N-src SH3 domain in a yeast two-hybridscreen. The src gene expresses an alternatively spliced form (N-src orpp60^(src-c)(*), which is specific for neuronal cells and has anincreased kinase activity (Brugge, et al., 1985). The N-src proteindiffers from the non-neuronal form (c-src or pp60^(src-c)) by aninsertion of six amino acids in the region corresponding to the Srchomology 3 (SH3) domain of the protein (Martinez et al. 1987). SH3domains are considered modules for protein-protein interaction (Pawson,et al., 1995). Therefore the yeast two-hybrid screen (Fields, et al.,1989; Zervos, et al., 1993) was used to identify brain specific proteinsthat would selectively interact with the N-src SH3 domain.

The screening of 5×10⁵ independent clones with the N-src SH3 baitresulted in the isolation of a single positively reacting fusion product(pJG-d5). This clone encoded a protein that showed a strong interactionwith the N-src SH3 domain, but no significant interaction with thec-src, fyn, or abl SH3 domains, indicating a specific recognition of theN-src SH3 domain in the yeast two-hybrid system. The sequence analysisof pJG-d5 indicated that it encodes the C-terminal portion of a largerprotein. Overlapping cDNA clones were therefore isolated from a λgt10library and an open reading frame (ORF) was identified that encodes a910 amino acid polypeptide with a predicted molecular weight of 104 kDa(FIG. 1A). The pJG-d5 insert corresponds to its C-terminal amino acids404-910.

The N-terminal part of the predicted protein contains an hydrophobiccore comprising seven hydrophobic domains (FIG. 1B). These domains showsignificant homology to the six transmembrane domains (S1-S6) and thepore region (P) of voltage activated K⁺ channels (FIG. 1C). In additionto the hydrophobic core, there is a putative cyclic nucleotide bindingsite (CNBs) in the C-terminal half of the protein (amino acids 472-602,FIG. 1A and FIG. 1D). This cyclic nucleotide binding site is mostclosely related to the corresponding region in cyclic-nucleotide gatedchannels (30% similarity). The amino acids that lie close to the boundcyclic nucleotide in the bacterial catabolite gene activator protein(CAP) are conserved in the N-src interacting protein, suggesting thatthe CNBs is functional (Weber et al., 1989). On the basis of thesefeatures, the newly identified protein was designated BCNG-1 (BrainCyclic Nucleotide Gated 1).

Among all the known K⁺ channel superfamily genes, the core region ofBCNG-1 displays the highest similarity (22%) to the corresponding regionin the mouse Eag protein, whereas the similarity to cyclicnucleotide-gated channels is only 17% in this region (distances weredetermined by the MegAlign program of DNASTAR). The S4 domain of BCNG-1has a total of eight positively charged residues (two groups of four,separated by a serine), which again makes it more similar to voltageactivated K⁺ channels (Sh and eag families) than to cyclicnucleotide-gated channels.

The putative pore forming region of the BCNG-1 protein (FIG. 1C) is alsomost closely related to the corresponding region in Shaker andEag-related channels (30% similarity in either case). However, itcontains significant substitutions in two positions that are otherwisehighly conserved in voltage activated K⁺ channels: the aspartate residuewhich follows the GYG triplet is replaced with alanine (position 352)and the serine/threonine residue at −8 from that position is replacedwith histidine (position 344). Similar substitutions are found in theβ-subunit of the retinal CNG-channel, where the position correspondingto the aspartate is occupied by a leucine and a lysine is found at −8from that position (Chen, et al., 1993). This suggests that the BCNG-1protein might be incapable of conducting current per se, but may act incombination with a second not yet identified polypeptide to form afunctional heteromultimeric ion channel.

BCNG-1 is a 132 kDa Glycoprotein. To characterize the protein encoded bythe BCNG-1 cDNA, antibodies were generated against two separate domainsin the predicted cytoplasmic tail: amino acids 594-720 (fusion proteinGST-q1; antiserum αq1) and amino acids 777-910 (fusion protein GST-q2;antiserum αq2). Both antisera specifically immunoprecipitated the invitro translation product of the cloned BCNG-1 sequence.

In Western blots of mouse brain extracts, both the αq1 and αq2 antiserarecognized a diffuse band with an apparent molecular mass of 132 kDa(FIG. 2A). Complete abolition of the labeling by preadsorbing theantisera with a GST-fusion protein incorporating both antigenic domains(GST-d5, amino acids 404-910) indicates it represents the native BCNG-1subunit. Treatment of the brain extract with N-glycosidase F prior tothe Western blotting results in a substantial reduction of the molecularweight of the observed band, which now co-migrates with the in vitrotranslated BCNG-1 product (FIG. 2B)

Sequence analysis indicates that three N-glycosylation consensus sitesare present in the BCNG-1 protein. Among these, Asn 327 is predicted tolie between transmembrane domain S5 and the pore (P) on theextracellular side of the plasma membrane (FIG. 1A and FIG. 1B). This,site corresponds to Asn 327 of the cGMP-gated channel from bovine rodphotoreceptors, where it has been demonstrated to be the sole site ofglycosylation (Wohlfart et al., 1992). Together, these data suggestedthat the cloned cDNA sequence encodes the full length product of theBCNG-1 gene and that BCNG-1 is a N-linked glycoprotein.

BCNG-1 is expressed in neurons. Northern blot analysis revealed thepresence of multiple BCNG-1 transcripts in poly(A)⁺ RNA from the brain,the most abundant species being 3.4, 4.4, 5.8 and 8.2 kb long (FIG. 3).The 3.4 kb transcript corresponds in size to the cloned cDNA. Noexpression was detected in the heart, spleen, lung, liver, skeletalmuscle, kidney or testis. The specific expression of the BCNG-1 proteinwas confirmed by Western blot analysis.

The cellular localization of BCNG-1 within the brain was examined by insitu hybridization (FIG. 4) and by immunohistochemical staining (FIGS.5A-5F). In both cases, the highest levels of BCNG-1 expression weredetected in the cerebral cortex, in the hippocampus, and in thecerebellum.

In the cerebral cortex, in situ hybridization shows a strong expressionof the BCNG-1 mRNA layer V pyramidal neuron cell bodies that aredistributed in a continuous line along the neocortex (FIG. 4).Immunohistochemical analysis reveals a strict subcellular localizationof the BCNG-1 protein within these cells. Staining of the apicaldendrites (FIG. 5A) extends into the terminal branches of these fibersand is particularly intense in layer I, which contains the terminaldendritic plexus of the pyramidal neurons (FIG. 5B).

A similar expression pattern can be recognized in the hippocampus. Here,the in situ hybridization shows a strong BCNG-1 mRNA expression in thepyramidal cell body layer of areas CA₁ and CA₃ (FIG. 4). The labeling inarea CA₃ is somewhat less prominent than the labeling in area CA₁. Atthe protein level, the most intense BCNG-1 immunostaining is observedalong the hippocampal fissure, in the layer corresponding to the stratumlacunosum-moleculare (FIG. 5C). This layer contains the terminalbranches of the apical dendrites of the pyramidal neurons in area CA₁(Raisman, 1965). Further BCNG-1 immunoreactivity is detected within thestratum pyramidale of areas CA₁ and CA₃; the staining, however, isabsent from the pyramidal cell bodies but is rather present in thefibers surrounding them (FIG. 5D). These fibers most likely representthe basket cell plexus associated to pyramidal neurons.

The immunostaining in the cerebellum also shows a pattern characteristicof basket cell expression. In the cerebellar cortex, basket cell nerveendings branch and contact the initial segment of the Purkinje cell axonin a distinct structure known as “pinceau” (Palay, et al., 1974). Asshown in FIGS. 5E and 5F, these structures are intensely labeled by theαq1 and αq2 antisera, while the staining excludes the Purkinje cellbodies. Thus, in basket cells, the BCNG-1 protein appears to beselectively localized to axons and is particularly enriched in the nerveterminals. An intense labeling of some brainstem nuclei is observed byin situ hybridization (FIG. 4) and areas of immunoreactivity weredetected in other brain regions, including the olfactory bulb.

BCNG-1 defines a new subfamily of K⁺ channel genes. Most of the ionchannel sequences characterized so far are members of evolutionarilyrelated multigene families. To investigate whether more sequencesrelated to BCNG-1 exist, mouse genomic DNA Southern blots were analyzedunder various stringency conditions (FIG. 6).

The probe (B1-T) was designed in the hydrophobic core region of BCNG-1,including transmembrane domains S5, P and S6; the repeat region in theC-terminal portion of the protein was excluded. Reducing the stringencyof the hybridization conditions from 8° C. below the melting temperatureof the B1-T probe (FIG. 6A) to 33° C. below the melting temperature(FIG. 6B) resulted in the detection of a number of additionalhybridization signals in every lane of the blot. None of the knownsequences in the K⁺ channel superfamily has sufficient homology toBCNG-1 to hybridize under these conditions. This result suggests thatBCNG-1 is the first known member of a larger group of related genes,which represent a new branch in the voltage-gated K⁺ channelsuperfamily.

Discussion

Voltage-gated potassium (VGK) channels constitute a large and stillexpanding superfamily of related genes (Strong, et al., 1993; Warmke andGonetzky, 1994). The most widely used strategy for cloning new genes inthe VGK family has been by homology to a small number of initial members(Sh, eag, and slo from Drosophila (Papazian, et al., 1987; Kamb, et al.,1987; Warmke, et al., 1991; Atkinson, et al., 1991); cGMP-channel frombovine retina (Kaupp, et al., 1989). Unfortunately, this approach is notwell suited for identifying more divergent sequences. Expression cloningin Xenopus ocytes can circumvent this problem, however, this implies apre-existing or readily detectable physiological characterization of thechannel.

An alternative cloning strategy that requires no a priori knowledge ofthe structure or activity of the target protein is to screen for K⁺channels by means of protein-protein interactions. Using the SH3 domainof N-src as a bait, a protein, mBCNG-1, was obtained that appears toconstitute a new branch of the K⁺ channel superfamily. mBCNG-1 displaysthe motifs of a voltage-gated K⁺ channel (six transmembrane spanningdomains, a highly basic S4, and a P region) (Strong, et al., 1993,Warmke, et al., 1994 and FIGS. 1A-1D). mBCNG-1, despite its similarityto voltage activated K⁺ channel superfamily members, with defined by thepresence of six transmembrane domains and a pore-like region (Warmke, etal., 1994), shows considerable divergence from all of the other knownsequences. Although the cyclic nucleotide binding site of BCNG-1 is mostsimilar to the site present in CNG channels (30%), the S4 and previousare most closely related to the corresponding regions in Shaker and Eag.Overall, the highest similarity in the hydrophobic core region is tomouse Eag Protein (22%). Thus, BCNG-1 appears to constitute a new branchof the K⁺ channel superfamily.

The fusion between an ancestral K⁺ channel and an ancestral cyclicnucleotide binding site is likely to have occurred prior to theevolutionary separation between plants and animals (Warmke, et al.,1994). Divergence from this common ancestor would have led on one handto Eag-related channels and plant inward rectifiers (which maintainedmore of the features of voltage activated K⁺ channels, while showing aprogressive deviation from the original CNBs sequence) and on the otherhand to CNG-channels (which show a higher evolutionary constraint on thecyclic nucleotide binding site, while they have lost voltage activationand K⁺ selectivity). The features of BCNG-1 suggest that it may haveremained closer to the ancestral molecule that represents theevolutionary link between voltage-gated K⁺ channels and cyclicnucleotide-gated channels.

The emerging pattern for olfactory and retinal CNG-channels and thenon-consensus sequence of the putative pore forming region of BCNG-1suggests that the lack of detectable electric current following BCNG-1expression in xenopus oocytes is due to BCNG-1 representing a β subunitof a heteromultimeric channel (Chen, et al., 1993; Liman, et al., 1994;Bradley, et al., 1994). Indeed the data show the existence of a numberof BCNG-related sequences in the mouse genome, and one or more of thesegenes could encode additional subunits required for the formation of anactive channel.

BCNG-1 protein is expressed only in the brain and in particular in twoof the principal classes of neurons within the cerebral, hippocampal andcerebellar cortexes: pyramidal neurons and basket cells. Thisdistribution would be consistent with an in vivo interaction of BCNG-1with N-src, which is also expressed in cerebral and hippocampalpyramidal neurons (Sugrue, et al., 1990). The observed interactionbetween BCNG-1 and the N-src SH3 domain is intriguing as is itsphysiological relevance and the role of the proline-rich region. Thepossibility that other factors may target the proline-rich region ofBCNG-1 has also to be considered, particularly in view of the recentlydiscovered WW domains (Sudol, et al., 1996; Staub, et al., 1996).

The varied subcellular localization of BCNG-1 (dendritic in pyramidalcells and axonal in basket cells) suggests that BCNG-1 could playdifferent roles in different populations of neurons, perhaps byregulating presynaptic or postsynaptic membrane excitability dependingon the cell type. A similar distribution has been demonstrated for theK⁺ channel subunit Kv 1.2 (Sheng, et al., 1994; Wang, et al., 1994). Kv1.2 forms heteromultimeric K⁺ channels with several other Shaker typesubunits, which have an overlapping yet differential pattern ofexpression, giving rise to a range of conductances with diversifiedfunctional characteristics.

The presence of BCNG-1 in the dendrites of hippocampal pyramidal cellsis particularly intriguing; cAMP has been shown to be important for theestablishment of some forms of long-term synaptic potentiation in thesecells (Frey, et al., 1993, Bolshakov, et al., 1997; Thomas, et al.,1996). The structural features of BCNG-1 predict a K+ conductingactivity, directly modulated by cyclic nucleotide binding.Interestingly, a current with similar characteristics has been describedin the hippocampal pyramidal neurons of area CA₁ (Pedarzani, et al.,1995), where BCNG-1 is highly expressed. This current (I_(Q)) isbelieved to contribute to the noradrenergic modulation of hippocampalactivity, by regulating neuronal excitability in response to cAMPlevels. BCNG-1 could participate in the formation of the channelsresponsible for this type of current.

Experimental Procedures

Yeast two hybrid interaction cloning of mBCNG-1. The two-hybrid screenwas performed following published procedures (Zervos, et al., 1993); thereagents used included plasmids pEG202, pJG4-5, pJK103 and Saccharomycescerevisiae strain EGY48 (MA Ta trp1 ura3 his3 LEU2:pLexAop6-LEU2).

The bait was created by subcloning the SH3 domain of N-src in plasmidpEG202, and contains amino acids 83-147 from the mouse N-src sequence(Martinez, et al., 1987). The cDNA fusion library was constructed inplasmid pJG4-5, using poly(A)⁺ RNA from the whole brain of an adultC57BL/6 male mouse; the cDNA was synthesized using random hexamers andthe GIBCO-BRL SuperScript II synthesis kit, according to themanufacturer's instructions. Only the library constructed from the twofractions with an average cDNA size of >1.5 kb (total of 1×10⁶independent clones) was used in the two hybrid screen. Libraryamplification was done in 0.3% SeaPrep agarose (FMC) to avoid changes incomplexity.

For library screening, Saccharomyces cerevisiae strain EGY48 was firstcotransformed with the bait plasmid pEG202-Nsrc and the reporter plasmidpJK103. The resulting strain was maintained under selection for the HIS3and URA3 markers, and subsequently transformed with the mouse brain cDNAlibrary in plasmid pJG4-5. This description though is more accurate andshould be substituted for the transformation mix was grown for two daysin a Ura⁻ His⁻ Trp⁻-glucose medium containing 0.3% SeaPrep agarose(FMC); the cells were then harvested and plated on Ura⁻ His⁻ Trp⁻Leu⁻-galactose. Leu⁺ colonies were screened for β-galactosidase activityusing a filter lift assay (Breede and Nasmith, 1985). Positivelyreacting fusion products were isolated and tested for specificityfollowing retransformation into an independent yeast strain. Fusionproduct pJGd5 corresponds to the C-terminal part of mBCNG-1 (amino acids404-910; see FIG. 8).

Full length cloning of mBCNG-1. For the isolation of the 5′ end regionof the mBCNG-1 cDNA, two rounds of PCR were performed on the pJG4-5library, using nested oligonucleotides derived from the pJG-d5 sequence.The downstream primer in the first round was:5′-AGAGGCATAGTAGCCACCAGTTTCC-3 (Seq. ID. No.: 13)(d5.RL, correspondingto amino acids 456-463 of the mBCNG-1 sequence; see FIG. 8). Thedownstream primer in the second round was:5′-CCGCTCGAGGCCTTGGTATCGGTGCTCATAG-3 (Seq. ID. No.: 14) (d5.N2),corresponding to amino acids 424-430 of mBCNG-1 and an added XhoI site).The upstream primer was either of two oligonucleotides designed in thepJG4-5 vector sequence: 5′-GAAGCGGATGTTAACGATACCAGCC-3′ (Seq. ID. No.:15) (B42), located 5′ to the EcoRI site in the B42 acidic patch, or:5-GACAAGCCGACAACCTTGATTGGAG-3 (Seq. ID. No.: 16) (ter), located 3′ tothe EcoRI site in the ADH terminator.

PCR cycling was performed as follows: 1×(2 minutes, 94° C.); 25× (45seconds, 94° C.; 30 seconds, 58° C.; 3 minutes, 72° C.); 1×(10 minutes,72° C.).

The longest amplification product obtained from this series of reactionswas a 700 bp DNA fragment, which contained amino acids 204-430 from themBCNG-1 sequence (See FIG. 8). This fragment was subcloned, repurifiedand used as a probe to screen a Mouse Brain cDNA library in λgt10(CLONTECH, cat. no. ML3000a), in high stringency conditions(hybridization overnight at 65° C. in 50% formamide, 5×SSC (1×SSC=0.15 Msodium chloride/0.015 sodium citrate, pH 7), 5× Denhardt's (1×Denhardt's 0.02% Ficoll/0.02% polyvinylpirrolidone/0.02% bovine serumalbumine), 0.5% SDS, 100 mg/ml salmon sperm DNA. Washing: 10 minutes,room temperature in 2×SSC/0.1% SDS, followed by twice 30 min at 65° C.in 0.2×SSC/0.1% SDS.

Positively reacting clones were further screened by PCR, usingoligonucleotide d5.RL (Seq. ID. No.: 13) as a downstream primer. Theupstream primer was either of the two following vector oligonucleotides:5 GAGCAAGTTCAGCCTGGTTAAGTCC-3 (Seq. ID. No.: 17) (15′.N2), located 5′ tothe EcoRI site in the λgt10 sequence, or 5′-GTGGCTTATGAGTATTTCTTCCAGGG-3(Seq. ID. No.: 18) (13′.N2), located 3′ to the EcoRI site. PCR cyclingwas performed as described above.

The resulting products were subcloned and sequenced. The longestextension contained amino acids 1-463 of the mBCNG-1 sequence (See FIG.8); the overlapping region of this insert with the insert contained inclone pJG-d5 (amino acids 405-463) includes a Bgl II site, which wasused to join the 5′ and 3′ fragments of the mBCNG-1 cDNA in plasmidpSD64TF for expression studies.

In vitro transcription (MESSAGE MACHINE, Ambion, Austin, Tex.) andtranslation in vitro Express, Stratagene.

Northern/Southern Blot Hybridization

PCR-generated cDNA fragments corresponding to the indicated amino acids6-131 (λgt10-derived) 5′ sequence) and 594-720 (pJG-5 derived 3′sequence) were used to probe a Multiple Tissue Northern Blot (CLONTECH,7762-1).

For Southern blots, a Mouse Geno-Blot (CLONTECH, 7650-1) was probedusing a PCR generated cDNA fragment (B1-T) corresponding to amino acids270-463 of the BCNG-1 sequence, as described (Sambrook, 1989). Blotswere hybridized at 65° (5× standard saline citrate 1×SSC=0.15M sodiumchloride/0.015 M sodium citrate, pH7 buffer in aqueous solution) andwashed as described in figure legends. Washings conditions were asindicated. The melting temperature (TM) for the B1-T probe wascalculated according to the formula Tm=81.5° C.+16.6 (logM)+0.41 (%GC)−(675/L), where M is the cation concentration and L is the probelength in base pairs.

Antibody production, extracts and Immunochemistry and in situhybridization. The Glutathione S-transferanse (GST)-fusion proteins werecreated by subcloning the q1 (corresponding to amino acids 594-720 ofthe mBCNG-1 protein) or q2 (corresponding to amino acids 777-910 of themBCNG-1 protein) (see FIG. 1A) in plasmid pGEX-lombole (Pharmacia),followed by induction and purification of essentially as described(Frangioni and Neel, 1993). Fusion proteins were eluted in phosphatebuffered saline (PBS) and injected into rabbits as a 1:1 suspension withFreund adjuvant (Pierce). Antisera were prepared and tested essentiallyas described (Grant, et al., 1995).

For Western Blot analysis, mouse brain extracts were separated on a 10%SDS-PAGE and electroblottted to PVDF membranes (Immobilon-P, Millipore)as described (Grant, 1995). Blocking and antibody incubations were donein TBST (10 mM Tris pH 7.5, 150 mM NaCl, 0.1% Tween-20)+2% BSA. The αq1and αq2 antisera were used at a 1:1000 dilution. Secondary anti-rabbitantibodies coupled to alkaline phosphatase (Bio-Rad) were used at a1:5000 dilution, and the bands were visualized by incubation in NBT\CIP(Boehringer Mannheim). Total brain extracts were prepared as described(Grant, 1995). For N-glycosidase treatment, 2% SDS was added to theextract and the proteins denatured by boiling for 10 min; reactions werecarried out in 50 mM NaP (pH 7.2), 25 mM EDTA, 0.5% Triton-X100, 0.2%SDS, 1 mg/ml protein and 20 U/ml N-glycosidase F (Boehringer) for 1 hrat 37C.

For immunohistochemistry, 20 μm cryostat sections of mouse brain (fixedin 4% paraformaldehyde/PBS), quenched in 50 mM NH4Cl/PBS, were blocked(10% goat serum, 0.1% goat serum, 0.1% saponin in PBS) and then exposedto αq1 or αq2 antisera (diluted 1:400 in blocking solution). Afterwashing in PBS+0.1% saponin, sections were incubated with Cy3-conjugatedgoat anti-rabbit F(ab′) 2 fragments (Jackson Immunoreasearch Labs)diluted 1:200 in blocking solution.

In situ hybridization was performed essentially as described (Mayford etal., 1995) using oligonucleotide probes labeled by 3¹ tailing withusing³⁵ (S) thio-dATP and terminal transferase (Boehringer Mannheim) toa specific activity of 5×10⁸ cpm/μg. Hybridizations were carried out at37° C. Slides were washed at 60° C. in 0.2×SSC and exposed to film for 2weeks.

Example 2 Initial Cloning and Description of the Mouse BCNG Gene Family

The original sequence in the BCNG family (mBCNG-1) was isolated from amouse brain cDNA library using yeast two-hybrid interaction cloning withthe n-Src tyrosine kinase as a bait as described in Example 1. The DNAand amino acid sequences of this protein are Seq. ID. No.:1 and Seq. ID.No.:2 respectively. Clones comprising two additional genes (mBCNG-2 andmBCNG-3) have been isolated. The DNA and amino acid sequences of mBCNG-2are Seq. ID. No.:5 and Seq. ID. No.: 6 respectively. The DNA and aminoacid sequences of mBCNG-3 are Seq. ID. No.:9 and Seq. ID. No.:10respectively. A more detailed description of the methods used toidentify the sequences reported here are given in the methods sectionbelow.

All of the identified sequences contain the motifs of a voltage gatedpotassium channel (six transmembrane spanning domains, including ahighly basic S4 domain, and a pore region) as well as a distinct cyclicnucleotide binding site (see FIG. 8). The three mouse proteins areclosely related to each other, having a similarity of 84-88%, but arevery distantly related to all other known members of the potassiumchannel superfamily, including Eag-related channels (22% similarity) andcyclic nucleotide-gated channels (17% similarity).

Northern blot analysis showed individual patterns of tissue distributionfor each of these clones (see FIG. 9). The expression of mBCNG-1 appearsto be restricted to the brain, whereas mBCNG-2 and mBCNG-3 are expressedin the brain as well as in the heart. Hybridization signals for mBCNG-3are also detected in polyA⁺ RNA from skeletal muscle and lung.

The distinct sequences and tissue distributions of these clones revealsthat the BCNG clones represent a family of ion channel proteins, withcharacteristic voltage sensing and cyclic nucleotide binding motifs,that are predominantly located in heart and brain.

Utilization of the EST data base in subsequent cloning of mouse andhuman BCNG genes. To assist in the complete cloning of the mouse genesand to begin the search for the human homologues of the BCNG genes, theEST data base was searched using the sequence of the mBCNG-1 protein.This search revealed four EST clones that appear to be fragments of twomouse BCNG genes (M41, M28) and two human BCNG genes (H57, H61).Correspondence between these ESTs and the BCNG gene family is shownschematically in FIG. 7 and is indicated in Table I. Clones wereobtained from the IMAGE consortium. The clones were used in thefollowing way.

M41-EST This sequence appeared to represent a 3′ region fragment of aBCNG-like gene, overlapping the cyclic nucleotide binding site. Anoligonucleotide was designed corresponding to the M41 sequence and wasused in an RT-PCR reaction together with an oligonucleotide designed ina conserved region of the 5′ portion of the BCNG clones (oligo B123(Seq.ID. No.: 23)). A product of the expected size was obtained, both frommouse brain and heart polyA⁺ RNA, subcloned and sequenced. An overlap inthe 5′ end region of M41-EST and in the 3′ end region of the partialclone of mBCNG-2 indicated that M41-EST represents the 3′ end region ofmBCNG-2. Accordingly, this clone is valuable to acquire the 3′ endregion of the gene for mBCNG-2. TABLE I EST CLONES IDENTIFIED BYHOMOLOGY TO mBCNG-1. GeneBank IMAGE Genome Trivial Probable Accessionconsortium systems Name Identity number cDNA ID ID M41-EST 3′ ofAA023393 456380 mBCNG-2 M28-EST 3′ of AA238712 693959 cd-22017 mBCNG-4H61-EST 3′ of N72770 289005 hBCNG-2 H57-EST 3′ of H45591 176364 hBCNG-1Table I list the trivial names (designated herein), the probablecorrespondence between these ESTs and the BCNG genes, the GeneBankaccession numbers and the clone identification numbers used by theI.M.A.G.E. consortium and Genome systems for these clones.

M28-EST. This clone also appears to contain a fragment of a BCNG-likegene, including the 3′ end region of the cyclic nucleotide binding site.An oligonucleotide to the M28 sequence was designed and used in anRT-PCR reaction together with the B123 oligonucleotide (Seq ID. No.:23). Again, a product of the expected size was obtained both from mousebrain and heart polyA⁺ RNA, subcloned and sequenced. This productappeared to represent an extension of mBCNG-3. However, no overlap waspresent with the M28-EST sequence, leading to the conclusion that M28represents yet another BCNG-like gene, which we designated BCNG-4.Indeed Northern blot analysis revealed a distinct pattern of tissuedistribution for BCNG-4 (see FIG. 9), which appears to be mainlyexpressed in the liver.

Complete sequencing of the M28-EST clone reveals that only the 3′ endregion of the clone aligns with the BCNG sequences; the sequence 5′ toposition 632 is likely to represent an intron (see Seq. ID. No.: 11 andSeq. ID. No.: 12 respectively).

H57-EST. Homology between H57-EST and mBCNG-1 suggested that this ESTmost likely represented the 3′ end region of hBCNG-1. The validity ofthis identification was strengthened by Northern blot analysis. FIG. 10shows that a probe constructed by PCR within the H57-EST sequence (seeFIG. 7 for probe details) recognized four transcripts in brain polyA⁺RNA from a Human Multiple Tissue Blot. This pattern is very similar tothat seen in the Northern blot of mBCNG-1 (see FIG. 9).

Based on this assignment, an oligonucleotide was generated correspondingto a sequence in the 5′ end region of mBCNG-1 and a secondoligonucleotide was designed within the H57-EST. Amplification yielded asingle, strong RT-PCR product of the predicted length from human brainPOLYA⁺ RNA. Complete sequencing of the original EST clone and of thereverse transcriptase-PCR (RT-PCR) product, together yielded 2247 bp ofthe hBCNG-1 sequence (Seq. ID. No.:3). The amino acid sequence is shownas Seq. ID. No.: 4. The core region of the hBCNG-1 proteins exhibits100% similarity to mBCNG-1.

H61-EST. This sequence showed marked sequence similarity to mBCNG-2 andmay represent the human homologue of the mBCNG-2 protein. Northern blotanalysis using a probe based on H61-EST (see FIG. 7) against a HumanMultiple Tissue Blot (FIG. 10) showed indeed an expression pattern whichis highly consistent with H61-EST being a 3′ region fragment of hBCNG-2.

Accordingly a sequence within the H61-EST was used to probe a humanbrain λgt10 cDNA library. Positive clones obtained from this screeningwere sequenced, and shown to encode the human homologue of the mBCNG-2protein. Together the sequences of the H61-EST and of the λgt10 clones,yielded 1792 bp of the hBCNG-2 sequence (Seq. ID. No.: 7). The aminoacid sequence is shown as Seq. ID. No.: 8). The core region of thehBCNG-2 protein is 98% homologous to mBCNG-2.

Functional expression studies of mouse and human BCNG gene familymembers. Because the BCNG genes appear to encode an ion channel, mBCNG-1was injected into in Xenopus oocytes as cRNA in order to recordcurrents, using both two electrode voltage clamp and excised inside-outpatch clamp approaches. A failure to detect current may result from afailure of correct trafficking or proper post-translationalmodification. In order to overcome such possible problems and to obtainfunctional expression of mBCNG-1, two approaches may be utilized.Firstly, expression of mBCNG-1 in a human cell line (HEK293 cells)and/or insect cell line (SF9 cells) may provide the properpost-translational modification. Second, expression in an insect cellsystem with a more efficient production of mBCNG-1 may yield afunctional ion channel. It is also possible that mBCNG-1 can not form afunctional protein alone and that it requires additional subunits. Thus,co-expression of other members of the BCNG family may circumvent thisproblem. Other members will be expressed individually and in combinationas they are cloned.

An alternative approach is to use other ion channel subunits that areknown to form heteromultimeric complexes and to co-express these witheach of the BCNG subunits proteins. An important guide to this approachis to establish first which proteins exhibit overlapping tissuedistribution with the BCNG proteins as determined from both Northernblot analysis (such as those in FIGS. 9 and 10) and immunochemicalapproaches as described herein.

Cloning of mBCNG-2. From the nested PCR reactions perform on the pJG4-5library (see Example 1, Full-length cloning of mBCNG-1) an amplificationproduct was isolated, that had a sequence similar but not identical tothe expected mBCNG-1 sequence. It was thus inferred that it representeda different gene, closely related to mBCNG-1, which we called mBCNG-2.The identified fragment encoded amino acids 234-430 from the mBCNG-2sequence (numbering according to mBCNG-1, see FIG. 8)

Next performed was a series of RT-PCR reactions on polyA⁺ RNA derivedfrom mouse brain and heart, using oligos: 5-TGGGAAGAGATATTCCACATGACC-3′(Seq. ID. No.: 19) (7.SEQ1, corresponding to amino acids 270-277 of themBCNG-1 sequence; see FIG. 8) as an upstream primer, and oligo d5.RL(Seq. ID. No.: 13) as a downstream primer. A 600 bp product was obtainedfrom heart polyA⁺ RNA, subcloned, sequenced and shown to be identical tomBCNG-2. PCR cycling: 1× (2 minutes, 94° C.); 25× (50 seconds, 94° C.;40 seconds, 52° C.; 1.5 minute, 72° C.); 1× (10 minutes, 72° C.).

The Clontech Mouse Brain λgt10 library was screened at high stringency(see Example 1, Full-length cloning of mBCNG-1), with a PCR probederived from the mBCNG-2 sequence (probe “dA”) using oligos:5′-TACGACCTGGCAAGTGCAGTGATGCGC-3′ (Seq. ID. No.: 20) (ASEQ2,corresponding to amino acids 278-286 of the mBCNG-2 sequence, numberingaccording to mBCNG-1; see FIG. 8) as an upstream primer, and5′-AGTTCACAATCTCCTCACGCAGTGGCCC-3′ (Seq. ID. No.: 21) (HRL.2,corresponding to aa 444-452 of the mBCNG-2 sequence, numbering accordingto mBCNG-1; see FIG. 8) as a downstream primer.

Positively reacting clones were further screened by PCR, usingoligonucleotide: 5′-CTGGTGGATATATCGGATGAGCCG-3′ (Seq. ID. No.: 22)(B2-ASE, corresponding to amino acids 262-269 of the mBCNG-2 sequence,numbering according to mBCNG-1; see FIG. 8) as a downstream primer andeither of the two lambda derived oligonucleotides (15′.N2 (Seq. ID. No.:17) or 13′.N2 (Seq. ID. No.: 18)) as an upstream primer (see example 1Full length cloning of mBCNG-1). The clones yielding the longestextension products were subcloned and sequenced, thus obtaining theN-terminal part of the mBCNG-2 sequence up to amino acids 304 (numberingaccording to mBCNG-1; see FIG. 8).

After obtaining the sequence for EST-M41, a further round of RT-PCRreactions was performed both on mouse brain and heart polyA⁺ RNA, usingoligonucleotides: 5′-CAGTGGGAAGAGATTTTCCACATGACC-3′ (Seq. ID. No.: 23)(B123, corresponding to aa 269-277 of the BCNG sequences, numberingaccording to mBCNG-1; see FIG. 8) as an upstream primer, and5′-GATCATGCTGAACCTTGTGCAGCAAG-3′ (Seq. ID. No.: 24) (41REV,corresponding to aa 590-598 of the mBCNG-2 sequence, numbering accordingto mBCNG-1; see FIG. 8) as a downstream primer. Extension products ofthe expected length were obtained from both RNA preparations, subclonedand sequenced, linking the λgt10 derived 5′ fragment and the EST derived3′ fragment of mBCNG-2.

PCR cycling was performed as follows: 1× (2 minutes, 94° C.); 25× (45seconds, 94° C., 30 seconds, 55° C.); 2 minutes, 72° C.); 1× (10 min,72° C.).

Cloning of mBCNG-3

From the λgt10 library screen for mBCNG-2 (see above) one positivelyreacting clone was obtained (#15) which appeared to give a consistentlyweaker hybridization signal. This insert was amplified witholigonucleotides 15.N2 (Seq. ID. No.: 17) and 13.N2 (Seq. ID. No.: 18),subcloned, sequenced and shown to represent a third BCNG-relatedsequence, different both from mBCNG-1 and mBCNG-2, which was calledmBCNG-3. The identified fragment encoded the N-terminal part of themBCNG-3 sequence up to aa 319 (numbering according to mBCNG-1; see FIG.8).

After obtaining the sequence for EST-M28, an RT-PCR was performed bothon mouse brain and heart polyA⁺ RNA using oligonucleotide B123 as anupstream primer, and degenerate oligonucleotide:5′-CACCKCRTTGAAGTGGTCCACGCT-3′ (Seq. ID. No.: 25) (28REV, correspondingto amino acids 554-561 of the BCNG sequences, numbering according tomBCNG-1; see FIG. 8) as a downstream primer. Extension products of theexpected length were obtained from both RNA preparations, subcloned andsequenced. Both represented extension of the mBCNG-3 sequence, asdetermined by an overlap with the known 3′ end of the λgt10 #15 clone.PCR cycling was performed at: 1× (2 minutes, 94° C.); 25× (45 seconds,94° C., 30 seconds, 55° C., 2 minutes, 72° C.); 1× (10 minutes, 72° C.).

Cloning of hBCNG-1. After obtaining the sequence for EST-H57, an RT-PCRreaction was performed on human brain polyA⁺ RNA, usingoligonucleotides: 5′-ATGTTCGGSAGCCAGAAGGCGGTGGAG-3′ (Seq. ID. No.: 26)(MB1-3, corresponding to aa 102-110 of the BCNG sequences, numberingaccording to mBCNG-1; see FIG. 8) as an upstream primer, and5′-CAGCTCGAACACTGGCAGTACGAC-3′ (Seq. ID. No.: 27) (H57.C, correspondingto amino acids 537-544 of the hBCNG-1 sequence, numbering according tomBCNG-1; see FIG. 8) as a downstream primer. A single extension productof the expected length was obtained, subcloned, sequenced, and shown torepresent the 5′ extension of the hBCNG-1 clone.

PCR was performed as follows: 1× (2 minutes, 94° C.); 25× (45 seconds,94° C., 20 seconds, 58° C., 3 minutes, 72° C.); 1× (10 minute, 72° C.).

Cloning of hBCNG-2. After obtaining the sequence for EST-H61, a PCRprobe was made using oligonucleotides: 5′AACTTCAACTGCCGGAAGCTGGTG3′(Seq. ID. No.: 28) (H61.A, corresponding to amino acids 452-459 of thehBCNG-2 sequence, numbering according to mBCNG-1; see FIG. 8) as anupstream primer, and 5′GAAAAAGCCCACGCGCTGACCCAG3′ (Seq. ID. No.: 29)(H61.F, corresponding to aa 627-634 of the hBCNG-2 sequence, numberingaccording to mBCNG-1; see FIG. 8) as a downstream primer on the EST-H61DNA. This fragment was used to screen a Human Brain Hippocampus cDNAlibrary in λgt10 (CLONTECH, cat. no. HL 3023a), in high stringencyconditions (see above). Positively reacting clones were further screenedby PCR, using oligonucleotide: 5′CACCAGCTTCCGGCAGTTGAAGTTG3′ (Seq. ID.No.: 30) (H61.C, corresponding to amino acids 452-459 of the hBCNG-2sequence, numbering according to mBCNG-1; see FIG. 8) as a downstreamprimer and either of oligonucleotides 15′.N2 (Seq. ID. No.: 17) or13′.N2 (Seq. ID. No.: 18) as an upstream primer. The clones yielding thelongest amplification products were subcloned and sequenced, thusobtaining the N-terminal region of the hBCNG-2 sequence up to aa 587(numbering according to mBCNG-1; see FIG. 8).

Northern blots. For mouse gene expression studies, a Mouse MultipleTissue Northern Blot (CLONTECH, cat. no. 7762-1) was probed with thefollowing PCR products: For mBCNG-1, probe “q0”, obtained using oligosq0.5′ (5′GCGAATTCAAACCCAACTCCGCGTCCAA3′) (Seq. ID. No.: 31) and q0.3′(5′CCTGAATTCACTGTACGGATGGAT3′) (Seq. ID. No.: 32). Amplification productcorresponding to aa 6-131 of the mBCNG-1 sequence (see FIG. 7 and FIG.8). For mBCNG-2, probe “dA”, obtained using oligos ASEQ2/HRL.2 (seeabove). For mBCNG-3, probe “15-7”, obtained using oligos 15.N2/13.N2(see above); amplification performed directly on lambda phage DNA (clone#15). For mBCNG-4, probe “M28” was obtained as a gel-purifiedEcoRI/BglII restriction fragment (400 bp) from the EST-M28 DNA. Fragmentcorresponding to amino acids 529-607 of the mBCNG-4 sequence (numberingaccording to mBCNG-1; see FIG. 8), plus 180 nucleotides of the mBCNG-43′UTR (untranslated region; see Seq. ID. No.: 11).

For human gene expression studies, a Human Multiple Tissue Northern Blot(CLONTECH, cat. no. 7760-1) or Human Brain Multiple Tissue Northern Blot(CLONETECH, cat. no. 7750-1) was probed with the following PCR products:For hBCNG-1, probe H57, obtained using oligos H57.A(5′GTCGTACTGCCAGTGTTCGAGCTG3′)(Seq. ID. No.: 33) and H57.B(5′GGTCAGGTTGGTGTTGTGAAACGC3′) (Seq. ID. No.: 34). Fragmentcorresponding to aa 537-800 of the hBCNG-1 sequence (numbering accordingto mBCNG-1; see FIG. 8). For hBCNG-2, probe “H61”, obtained using oligosH61.A (Seq. ID. No.: 28) and H61.F (Seq. ID. No.: 29)(see above).

Hybridizations were all performed in ExpressHyb solution for 1 hour, 68°C., as indicated in the manufacturer's Protocol Handbook. Washing wasperformed as follows: 10 minutes, room temperature in 2×SSC/0.1% SDS,followed by twice 30 minutes, 65° C. in 0.2×SSC/0.1% SDS. Filters werestripped between subsequent hybridizations by boiling for 5 min in 0.5%SDS/H₂O.

Sequence alignments and EST database search. Alignments and distancecalculations were all performed with MegAlign (DNASTAR) on the indicatedpeptide sequences.

The EST database search was performed with BLAST (NCBI), using themBCNG-1 polypeptide sequence (amino acids 1-720, to avoid the glutaminerepeat present in the C-terminal region of the protein) and the TBLASTNprogram.

Discussion

Ion channels are important targets of drugs for treatment of a varietyof neurological and cardiovascular diseases. Members of the novel BCNGfamily of ion channels are expressed at the mRNA level in brain, cardiacmuscle sketch muscle, lung, kidney and liver. From their amino acidsequence, these channels described herein are likely to have twoimportant physiological properties that make them a priori attractivetargets for drug development. First, they are members of thevoltage-gated channel family and are most similar to voltage-gated K⁺channels. Second, they possess a cyclic nucleotide binding domain intheir carboxy terminus, suggesting that they will be directly regulatedby cyclic nucleotides. Based on these properties, combined with thetissue distribution and high levels of expression at mRNA levels, thesechannels are likely to play important roles in controlling neuronal andcardiac electrical activity. The regulation of these channels throughdrugs may as well as transport functions in lung, liver and kidneyprovide a unique opportunity for regulating electrical activityassociated with diseases as diverse as epilepsy, cardiac arrhythmias andcystic Fibrosis. Moreover, the cyclic nucleotide binding domain of thesechannels provides a unique pharmacological target that could be used todevelop novel, specific, cyclic nucleotide agonists or antagonists toupregulate or downregulate channel function.

Finally, these channels are attractive candidate genes for the pacemakercurrent that underlies spontaneous activity in the heart and variousnuclei in the brain. This hypothesis is based on physiological studieswhich have shown that pacemaker channels are expressed in both heart andbrain and have the unique property of being gated by both voltage andthe direct binding of cyclic nucleotides to a cytoplasmic site on thechannel—properties similar to those predicted for BCNG channels. Asthese pacemaker channels regulate the rhythmic beating of the heart,respiration, attention, sleep and wakefulness, they are prime targetsfor drug therapy. The expression of BCNG gene family members inheterologous systems, such as mammalian cell lines, facilitates rapiddrug screens that could selectively identify compounds that targetchannel subunits encoded by BCNG genes expressed in brain or heart.

Example 3 Physiological and pharmacological significance of mouse andhuman BCNG channel genes.

Introduction

The unique structural features and the tissue distribution of thepredicted proteins of the BCNG gene family suggests that they may encodethe pacemaker current (variously called Ih, If or Iq) of the heart andbrain. Even if the BCNG gene family does not encode the pacemakercurrent it is likely to be a component of other—perhapsunidentified—ionic current(s) that are important in cardiac renal,hepatic and central nervous system function. Irrespective of theidentity of the current(s) that the BCNG proteins contribute to, theunique structural features of the predicted BCNG proteins (the unusualion conducting pore (P) domain, the highly conserved cyclic nucleotidebinding (cnbs) site and the highly conserved and highly charged S4voltage sensor) indicate that they will be susceptible to multiple drugintervention strategies that target the pore, the cyclic nucleotidebinding site and the voltage-dependent gating apparatus.

Analysis And Predicted Structure and General Features of the BCNGProteins. The predicted amino acid sequence of the BCNG genes revealsthat they are members of the voltage-gated ion channel superfamily.Specifically, the BCNG proteins show similarities to the superfamily ofchannels that includes the voltage-gated K⁺ channels (Pongs, et al.,1995) and the cyclic nucleotide-gated channels, non-selective cationchannels that are permeable to Na, K⁺ and Ca (Zagotta and Siegelbaum,1996). As shown schematically in FIG. 11, the BCNG proteins arepredicted to have six transmembrane spanning a-helices with cytoplasmicN and C termini, a highly basic four transmembrane domain (S4) and pore(p) region. Each these motifs are found in the members of thevoltage-gated K⁺ family. In addition, the BCNG proteins have a wellconserved cyclic nucleotide binding site in the C-terminus. Although ahomologous motif is found in some of the voltage-gated K⁺ channels, thecyclic nucleotide binding sites in those channels are not well conservedand there is little evidence that the binding sites are functional.Indeed, the cyclic nucleotide binding site of the BCNG channels is mosthomologous to the sites found in cyclic nucleotide gated channels whichuse the binding of cyclic nucleotides to drive their activation.Furthermore, while the P loops of the BCNG channels are homologous tothose found in voltage activated K⁺ channels and cyclic nucleotide gatedchannels, there are several non-conservative changes in the amino acidsequence that are likely to yield ion conduction properties that areunique to the BCNG channels. Thus, the BCNG channels appear distinctfrom all previously identified channels in a number of ways whichsuggests that they have distinct physiological and pharmacologicalproperties. These similarities and dissimilarities in the sequences ofthe voltage-gated K⁺ channels, cyclic nucleotide-gated channels and theBCNG channels and the predicted consequences for BCNG channel functionalproperties are discussed more extensively below.

Results

The Hydrophobic Core. The core of BCNG channels is predicted to have sixtransmembrane (α-helical sequences (S1-S6) and a pore forming P loop.This assignment is homologous to a single subunit of the tetrameric K⁺channels (and tetrameric cyclic nucleotide-gated channels) or singlerepeat in the pseudo tetrameric Na and Ca channels. This homologysuggests that the BCNG channels are members of the voltage-gated K⁺channel superfamily (which also includes the voltage-independent butstructurally homologous cyclic nucleotide-gated channels). It is likelythat the BCNG channels will be composed of four such polypeptides in ahetero or homomultimeric structure as is seen for the voltage-gated K⁺channels and the cyclic nucleotide-gated channels (Chen et al., 1993;Bradley et al., 1994; Liman and Back, 1994; Lin et al., 1996). However,BCNG-1 shows considerable divergence from all other known K⁺ channel andcyclic nucleotide-gated channel sequences. As noted above, the highesthomology in the hydrophobic core region is to mouse Eag (22% amino acidsimilarity)—a voltage-gated K⁺ channel that has a degenerate andprobably non-functional cyclic nucleotide binding site Warimke andGancleky. Over this core region, mBCNG-1 shows 17% identity to thevoltage independent cyclic nucleotide-gated channels.

In contrast, the proteins that are predicted to be encoded by the BCNGgenes show high homology to each other (>80%, see Examples 1 and 2).Indeed, mouse BCNG-1 and human BCNG-1 are identical over the coreregion. Similarly, mBCNG-2 and hBCNG-2 are 98% identical over the coreregion. Thus, the BCNG family of genes appears to constitute a newbranch of the K⁺ channel superfamily which could be regulated by cyclicnucleotide binding. The presence of a gene family with members showingsuch sequence conservation strongly suggests important biologicalfunction.

The S4 voltage-sensing domain. The presence of positively chargedarginine and lysine residues at every third posit in the fourthtransmembrane helix is a signature sequence voltage-dependent gating(Hille, 1992; Catterall, 1992, see FIG. 12). In contrast, in thevoltage-independent cyclic nucleotide-gated channels, the S4 isdegenerate with some of the positively charged residues being replacedby negatively charged acidic amino acids or being out of the triadrepeat frame. These changes have reduced the net positive charge in theS4 of the cyclic nucleotide-gated channels to 3-4. This introduction ofnegatively charged residues may underlie the reason that the cyclicnucleotide-gated channels no longer respond to voltage. However, it isalso possible that voltage-sensitivity may have been lost as a result ofsome other structural change in the cyclic nucleotide-gated channels andthe divergence in S4 structure is simply a reflection of the loss ofevolutionary pressure to retain the positive charges.

The S4 of BCNG channels are most closely related to the correspondingregions in the voltage-gated K⁺ channels Shaker and eag, albeit poorly(mBCNG-1 is 30% homologous to the S4 of Shaker and eag). Despite aninterruption by the inclusion of a serine in place of an arginine in theS4 of BCNG channels, the BCNG sequence contains more positively chargedresidues than any other member of the voltage-gated K⁺ channelsuperfamily (see FIG. 12). The S4 domain of BCNG-1 has up to ninepositively charged residues (one group of five and one group of fourseparated by a serine in place of one other arginine), which again makesit more similar to voltage activated K⁺ channels (Sh and eag families)than to cyclic nucleotide-gated channels. The retention of such a highlycharged S4 strongly suggests that the gating of these channels arevoltage-sensitive.

The cyclic nucleotide binding site. Cyclic nucleotides regulate theactivity of a diverse family of proteins involved in cellular signaling.These include a transcription factor (the bacterial cataboliteactivating protein, CAP), the cAMP-and cGMP-dependent protein kinases(PKA and PKG) and the cyclic nucleotide-gated (CNG) ion channelsinvolved in visual and olfactory signal transduction (Shabb and Corbin,1992; Zagotta and Siegelbaum, 1996). Despite obvious divergence amongthe effector domains of these proteins, the cyclic nucleotide bindingsites appear to share a common architecture. Solution of the crystalstructures of CAP (Weber and Steitz, 1987) and a recombinant bovine PKAR1α subunit (Su, et al., 1995) has demonstrated that their cyclicnucleotide binding sites are formed from an α helix (A helix), an eightstranded β-roll, and two more α-helices (B and C), with the C-helixforming the back of the binding pocket. Of the approximately 120 aminoacids that comprise one of these cyclic nucleotide binding sites, sixare invariant in all CAP, PKA, PKG and cyclic nucleotide gated channels.Thus, it has been suggested that the invariant residues playimportant—and conserved—roles in the folding and/or function of the CNBsites of these diverse proteins (Shabb and Corbin, 1992; Zagotta andSiegelbaum, 1996; Weber and Steitz, 1987; Su, et al., 1995; Kumar andWeber, 1992; Scott, et al., 1996). Indeed, the crystal structure of CAP(Weber and Steitz, 1987) and the regulatory subunit of recombinantbovine R1α (Su, et al., 1995) reveals that the glycines are at turnswithin the β-roll while the glutamate and the arginine form bonds withthe ribose-phosphate of the nucleotide.

Interestingly, only three of these residues—two glycines and thearginine—appear to be conserved among the more distantly relatedvoltage-gated channels which bear the CNB site motif but whose gatingmay NOT be modulated significantly by direct binding of cyclicnucleotide (KAT1 (Hoshi, 1995) and drosophila EAG (dEAG) (Bruggeman, etal., 1993) (see FIG. 11).

Thus in the plant channel, KAT1, the first glycine is mutated to anasparagine and the alanine is changed to a threonine. In dEAG theglutamate in changed to an aspartate. Furthermore, the alignment of dEAGto the highly conserved RXA sequence in β-7 is uncertain. Often, the SAAsequence within the dEAG β-7 is aligned with the RXA consensus sequencewhich suggests that the arginine is lost and replaced with a serine. RALis aligned with the RXA consensus sequence which would indicate that thearginine is retained but the alanine is replaced with a leucine.Regardless of which alignment is considered, it is clear that thebinding site sequence of KAT1, dEAG and related channels all showdeviations from the consensus motif for a functional cyclic nucleotidebinding site. In keeping with this structural divergence, there is onlyone report that any cloned EAG is being sensitive to direct cyclicnucleotide binding (Bruggermann et al., 1993). However, this result hasnot been confirmed and it is now thought to be an artifact. There issome evidence that the gating of the plant channel KAT1, may be weaklysensitive to cyclic nucleotide modulation (Hoshi, 1995).

FIG. 13 shows a schematic representation of the cyclic nucleotidebinding site of bRET1 showing the critical interactions between thebinding site and the cyclic nucleotide.

Recent evidence has demonstrated that the third (or C) α-helix movesrelative to the agonist upon channel activation, forming additionalfavorable contacts with the purine ring. Indeed, the selectiveactivation of bRET1 by cGMP relative to cAMP is largely determined byα-residue in the C α-helix, D604 {Varnum et al., 1995}. This acidicresidue is thought to form two hydrogen bonds with the hydrogens on aring nitrogen and amino group of the cGMP purine ring. Unlike the cGMPselective bRET1 channel, cyclic nucleotide-gated channels that areactivated equally well by cAMP or cGMP (fOLF1, Goulding et al., 1992;Goulding et al., 1994) or which favor activation by cAMP (rOLF2coexpressed with rOLF1, Liman & Buck, 1994; Bradley et al., 1994) do nothave an acidic residue here, but rather, have polar or hydrophobic aminoacids (see Varnum, et al., 1995}. Neutralization of D604 results in aloss of the ability to form the favorable hydrogen bonds with cGMP andthe loss of the unfavorable interaction with the lone pair of electronson the purine ring of cAMP, thus accounting for the channels which beara hydrophobic or polar residue at this position becoming non-selectivebetween cAMP and cGMP or even selective for cAMP.

In the C-terminus of the BCNG proteins there is a sequence ofapproximately 120 amino acids that is homologous to these cyclicnucleotide binding sites. Strikingly, the six residues that have beenshown to be totally conserved in all functional cyclic nucleotidebinding sites are conserved all of the BCNG proteins that we haveidentified so far. The cyclic nucleotide binding site of the BCNGchannels are most similar to the functional site present in thevoltage-independent cyclic nucleotide-gated channels (30%). When thecyclic nucleotide binding sites found in channel genes are compared tothose in protein kinases, the BCNG channel sites are more similar (25%similarity to yeast cAMP-dependent protein kinases) than those of anyother ion channel. These data strongly suggest that the BCNG genesencode proteins whose activity is modulated by direct binding of cyclicnucleotide. Furthermore, the BCNG channels all have an isoleucineresidue in the position where D604 is found in the cGMP selective bRET1channel. Thus, the BCNG are probably cAMP selective.

The pore. Despite the functional divergence that has given rise to Na,Ca or K selective families and to the presence of channels within thesefamilies whose conductances vary by 1-2 orders of magnitude, the poresof all members of the voltage-gated superfamily are related (see Itillic1992; For example see FIG. 14). Much is known about the residues thatcontribute to the ion permeation properties of channels and this allowspredictions about the permeation properties of the BCNG proteins(Mackinnon, 1991; Heginbothem et al., 1994).

Overall, the P region of mBCNG-1 is most closely related to thecorresponding region in the K selective Shaker and eag channels, albeitpoorly (30%). Based on the presence of a GYG motif in the P loop, theBCNG proteins would be expected to be K selective. However, the BCNG Ploops contain substitutions in several positions that are otherwisehighly conserved in other voltage activated K+ channels. These changescan be seen in FIG. 14 (which shows an alignment of mBCNG-1 againstchannels from all the other major K channel families) and FIG. 8 (whichshows the alignment of all currently cloned BCNG sequences). Theaspartate residue which follows the GYG triplet is replaced with alanine(position 352) in mouse and human BCNG-1 and by an arginine in the otherBCNG channels identified so far. The serine/threonine residue 8 residuesN-terminal from that position (residue 344 in mBCNG-1) is replaced withhistidine in all of the BCNG sequences. 6 residues N-terminal from theaspartate a hydrophobic leucine residue is introduced in place of apolar residue. In addition, at position −12 from the aspartate, a sitethat is occupied by an aromatic residue in all of the other channelsaligned in FIG. 14, a lysine residue is introduced in all of the BCNGsequences (FIG. 8 and FIG. 14).

Interestingly, some of the substitutions found in the BCNG channels aresimilar to those found in the β-subunit of the CNG-channel (see hRET-2in FIG. 14). In hRET-2, the position corresponding to the aspartate isoccupied by a leucine, while a lysine is found at −8 from that positionand an isoleucine is found at position −6 (Chen et al., 1993). Thisobservation suggests that the BCNG proteins cloned so far might be the1-subunits of the BCNG channel family and the α-subunits have yet to beidentified. Indeed, a failure to observe an ionic current withhomomerically expressed mBCNG-1 is reminiscent of the cyclic nucleotidegated family 1-subunits {Liman & Buck, 1994; Bradley et al., 1994} andthe γ-subunits of the voltage-gated Shal K channel {Jegla & Salkoff,1997}. When first cloned, the cyclic nucleotide-gated channel β-subunitswere thought to be incapable of forming functional channels in theabsence of the homologous α-subunits and that their normal physiologicalrole was to generate a greater diversity of channel phenotypes byheteromeric assembly with the α-subunits. While it is clear that boththe cyclic nucleotide-gated family β-subunits and the voltage-gated Kchannel γ-subunits can form heteromultimers with their respectiveα-subunits, it has now been demonstrated that the cyclicnucleotide-gated channel β-subunits can form homotetrameric channelsthat can be activated by nitric oxide (NO) modification of a cytoplasmiccysteine residue {Broillet & Firestein, 1997}. Although thephysiological significance of this is uncertain and homomeric expressionof the γ-subunits of Shal has not yet been demonstrated we are going toexplore the possibility that the present family of BCNG channel subunitsneed additional subunits to form functional channels.

Although the amino acid substitutions seen in the P region of the BCNGsubunits do not necessarily indicate that the channel will have lost itsK selectivity (for example a lysine is present in the P region of the Kselective Shaw channel, See FIG. 14) the substantial deviations from theK channel consensus sequence suggest that the BCNG proteins may generatea family of channels that do not select well between Na and K—consistentwith the hypothesis that the BCNG channels encode the non-selective Ihcurrent.

Discussion

Significance of the BCNG Structure

The presence of cyclic nucleotide binding sites on a number of K⁺channels that are found in both plant and animal phyla suggests that thefusion between an ancestral K⁺ channel and an ancestral cyclicnucleotide binding site is likely to have occurred prior to theevolutionary separation between plants and animals (Warmke and Ganetzky,1994). Indeed, the finding that many of these sites are degenerate andnon-functional supports this interpretation. Divergence from this commonancestor would have led on one hand to Eag-related channels (EAG, ERG,ELK) (Warmke and Ganetsky, 1994) and plant inward rectifiers (AKT andKAT), which maintained more of the features of voltage activated K⁺channels, while showing a progressive deviation from the original cyclicnucleotide binding site sequence) and on the other hand to CNG-channels(which show a higher evolutionary constraint on the cyclic nucleotidebinding site, while they have lost voltage activation and K⁺selectivity) (Anderson, et al., 1992; Sentenac, et al., 1992). Thefeatures of BCNG-1 suggest that it may have remained closer to theancestral molecule that represents the evolutionary link betweenvoltage-gated K⁺ channels and cyclic nucleotide-gated channels. This issupported by the observations that the cyclic nucleotide binding site ofmBCNG-1 shows the closest homology to binding sites present in proteinkinases, in particular in yeast cAMP-dependent protein kinases (25%)while the channel domains are most closely related to thevoltage-dependent channel encoded by the Shaker gene that does not havea cyclic nucleotide binding site and thus, presumably arose before thegene fusion event. The cyclic nucleotide binding site of mBCNG-1 is mosthomologous to the site present in cyclic nucleotide-gated channels (30%)which again demonstrates that these probably arose from a commonancestor and in both there was pressure to maintain the cyclicnucleotide binding site because it contributed to the function of theprotein. Thus, BCNG-1 appears to constitute a new branch of the K⁺channel superfamily.

Physiological Significance. Ion channels are central componentsunderlying the electrical activity of all excitable cells and serveimportant transport functions in non-excitable cells. Members of thenovel BCNG family of ion channels are expressed in both brain andcardiac muscle as well as skeletal muscle, lung, liver and kidney. Fromtheir amino acid sequence, members of the BCNG channel gene family arelikely to have important, novel roles in the electrophysiologicalactivity of the brain and the heart and other tissues. This view isbased, first, on the finding that mRNA coding for BCNG channel proteinis expressed in both heart and brain. Second, the deduced primary aminoacid sequence of the BCNG channels indicate that they are members of thevoltage-gated channel family but unlike most voltage-gated channels, theBCNG channels contain what appears to be a functional cyclicnucleotide-binding domain in their carboxy terminus.

Northern blots of the four mouse BCNG channel genes show interestingdifferences in expression patterns (see FIG. 3, FIG. 9 and FIG. 10).mBCNG-1 is selectively expressed in brain. Western blots confirm thatmBCNG-1 is also highly expressed at the protein level, and that thisexpression is widespread throughout the mouse brain (see FIG. 2).mBCNG-2 is expressed in brain and heart. mBCNG-3 is expressed in brain,heart, lung and skeletal muscle. mBCNG-4 is expressed in brain, liverand kidney. Thus, each gene, although highly similar at the amino acidlevel, shows a distinct pattern of expression, implying that each has aunique physiological function. This is borne out by the finding that thetwo human members of the BCNG family, hBCNG-1 and hBCNG-2, show similarpatterns of expression to the mouse homologs. Thus, hBCNG-1 isselectively expressed in brain whereas hBCNG-2 is expressed in brain andheart. Even within a particular organ system, the different genes showdifferent patterns of expression. Thus, in the brain hBCNG-1 is morehighly expressed in hippocampus and amygdala than in other brainregions. In contrast, hBCNG-2 is highly expressed in all brain regions.

Based on the BCNG amino acid sequence and tissue distribution, it ishypothesized that the channels encode a either a voltage-gated potassiumchannel that is activated by membrane depolarization and modulated bythe direct binding of cyclic nucleotides, or thehyperpolarization-activated pacemaker channel that underlies spontaneouselectrical activity in the heart DiFrancesco and Torta, 1991 and invarious regions of the brain Steride et al., 1993. This latterhypothesis is based on the finding that the pacemaker channels, similarto BCNG genes, are expressed in both brain and the heart. Moreover, thepacemaker channels are known to be non-selective cation channels thatare gated by both voltage and the direct binding of cyclic nucleotidesto a cytoplasmic site on the channel (DiFrancesco and Torta, 1991;Pedarzani and Storm, 1995 Larkman And Kelly, 1997, McCormick and Page,1990). To date, there is no biochemical or molecular biologicalinformation as to the nature of the pacemaker channel. However, thesimilarity in tissue distribution and proposed gating mechanisms betweenthe pacemaker channels and the BCNG channels suggests that the BCNGgenes code for one or more subunits that comprise the pacemakerchannels.

Pacemaker channels have been studied at the electrophysiological levelin both cardiac tissue and central neurons. In both instances, thechannels are activated when the cell membrane voltage is made morenegative than −40 mV. These non-selective channels are permeable to bothNa and K⁺ ions. However, at the negative membrane potential range overwhich these channels open, their main effect is to allow positivelycharged sodium to enter the cell from the extracellular environment,causing the cell membrane to become more positive. This eventuallycauses the membrane voltage to reach threshold and the cell fires anaction potential. (See FIG. 15). Cyclic AMP (cAMP) is known to shift therelation between membrane voltage and channel activation, causing thechannels to turn on more rapidly when the membrane is depolarized. Thisincreases the rate of the pacemaker depolarization, increasing the rateof spontaneous action potential firing. It is this effect that underliesthe ability of epinephrine (adrenaline) to cause the heart to beatfaster. The effects of cAMP on the pacemaker current appear to occurthrough two separate molecular mechanisms. First, cAMP activates theenzyme, cAMP-dependent protein kinase (PKA), leading to an increase inlevels of protein phosphorylation. Second, cAMP is thought to directlybind to a cytoplasmic region of the pacemaker channel, producing aneffect similar to that seen with protein phosphorylation. Such directactions of cAMP have been reported both in the heart and in br(DiFrancesco and Torta, 1991; Pedarzani and Storm, 1995,

An alternative function for the BCNG channels, that they encode for anovel voltage-gated and cyclic nucleotide-gated potassium channel, issuggested by the amino acid region that is known to line theion-conducting pore and hence determine the ionic selectivity of thechannel. This S5-S6 loop contains a three amino acid motif, GYG, that isconserved in almost all voltage-gated K⁺ channels Heginbotham et al.,1994. The BCNG channel S5-S6 loop shows amino acid similarity with thatof other potassium channels, including the GYG motif. This suggests thatthe BCNG channels may be K⁺ selective. However, there are a number ofstriking differences in the sequence between BCNG and other K⁺ channelsthat may indicate that the BCNG channels are less K-selective comparedto other K⁺ channels, consistent with the view that the BCNG channelscode for the non-selective cation pacemaker channels that are permeableto both Na and K⁺.

The presence of BCNG-1 in the dendrites of hippocampal pyramidal cellsis particularly intriguing, as cAMP has been shown to be important forthe establishment of some forms of long-term synaptic potentiation inthese cells (Frey, et al., 1993; Boshakov, et al., 1997; Huang andKandel, 1994; Thomas, et al., 1996).The structural features of BCNG1predict a K⁺ conducting activity, directly modulated by cyclicnucleotide binding.

Interestingly, a current with similar characteristics has been describedin the hippocampal pyramidal neurons of area CAl (Warmke, et al., 1991)where BCNG-1 is highly expressed. This current (IQ) is believed tocontribute to the noradrenergic modulation of hippocampal activity, byregulating neuronal excitability in response to cAMP levels. BCNG-1 mayparticipate in the formation of the channels responsible for this typeof current.

By analogy with the emerging pattern for olfactory and retinalCNG-channels, the non-consensus sequence of the putative pore formingregion of mBCNG-1 suggests that this protein may represent a 1-subunitof a heteromultimeric channel (Liman and Buck, 1994; Bradley, et al.,1994). Indeed the data show the existence of a number of BCNG-relatedsequences in the mouse genome, and one or more of these genes couldencode additional subunits required for the formation of an activechannel. These channels are likely to have two important physiologicalproperties that suggest they serve important functions in the role ofmany excitable cells. First, they are members of the voltage-gatedchannel family and are most similar to voltage-gated K⁺ channels.Second, they possess a cyclic nucleotide binding domain in their carboxyterminus, suggesting that they will be directly regulated by cyclicnucleotides. Based on these properties, combined with their tissuedistribution and high levels of expression at the mRNA levels, thesechannels are likely to play important roles in controlling neuronal andcardiac electrical activity as well as having important functions inother excitable and non-excitable cells.

Based on the widespread tissue distribution and likely importantphysiological role of the BCNG channels in electrical signaling, drugsthat interact with these channels are of potential therapeutic use in anumber of neurological, psychiatric and cardiac diseases as well assystemic diseases of tissues such as skeletal muscle, liver and kidney.

Neurological disease: Based on the high expression of these channels inthe hippocampus and potential role in spontaneous pacemaker activity,they may be useful, novel targets for treatment of epilepsy. Forexample, by blocking these channels it may be possible to prevent ordiminish the severity of seizures. In diseases associated withhippocampal neuronal loss, such as age-related memory deficit,stroke-induced memory loss, and Alzheimers disease, a drug whichenhanced pacemaker channel activity may be of therapeutic use byincreasing neuronal activity in the hippocampus. As these channels arealso expressed in the basal ganglia and striatum, they may be potentialtargets in Parkinson's and Huntington's disease. The BCNG channels arealso highly expressed in the thalamus, where pacemaker channels havebeen shown to be important in generating spontaneous action potentialsimportant for arousal. Targeting of such channels may help treatattention deficit disorder.

Psychiatric disease: Given the high levels of expression of hBCNG-1 inthe amygdala, these channels may be targets for drugs involving variousaffective disorders and anxiety. Their high expression in the limbicsystem suggests that they may also be of potential benefit in treatmentof schizophrenia.

Cardiac disease: The expression of the BCNG-2 channels in the heartsuggests that they may be useful targets for treatment of certaincardiac arrhythmias. Based on the hypothesis that these genes may encodepacemaker channels the BCNG channels will be potential targets fortreats both bradyarrhythmias through drugs that enhance pacemakerchannel activity and certain tachyarrhythmias due to enhancedautomaticity. Even if the BCNG channels are not the pacemaker channels,they are likely to play an important role in cardiac electricalactivity, perhaps contributing to action potential repolarization, andthus would remain attractive targets for drug development.

A number of drugs, toxins and endogenous compounds are known to interactwith various types of ion channels. These drugs have proved useful aslocal anesthetics and in the treatment of cardiac arrhythmias,hypertension, epilepsy and anxiety. These drugs fall into severalclasses including pore blockers, allosteric modulators, and competitiveantagonists (see Table II). The BCNG channels present some uniquefeatures that make them very attractive drugs. First, there are bothbrain specific genes (BCNG-1) and genes that expressed in both brain andheart (BCNG-2,3). Thus BCNG may yield drugs specific for brain or heart.Second, the pore region of the BCNG channels shows considerabledivergence from that of other known potassium channels. Thus, yieldingpore-blocking drugs that would selectively alter BCNG channels but spareother types of voltage-gated K⁺ channels. Third, the cyclicnucleotide-binding site elucidate another important target with respectto the opening of the BCNG channels. By designing specific cyclicnucleotide analogs it should be possible to design either syntheticagonists, which will increase channel opening, or antagonists which willdecrease channel opening. Most available drugs for ion channels decreasechannel opening, relatively few increase channel opening. The ability toeither increase or decrease the opening of BCNG channels offers muchpotential for therapeutically effective compounds. For example in bovinephotoreceptor CNG channels, Rp-cGMPS is an antagonist of channel openingwhereas Sp-cGMPS is an agonist (Kramer and Tibbs, 1996). Moreover, theamino acid sequence of the cyclic nucleotide binding site of the BCNGchannels shows considerable divergence with cyclic nucleotide bindingsites of protein kinases and the cyclic nucleotide-gated channels ofolfactory and photoreceptor neurons. Thus it should be possible todesign cyclic nucleotide analogs which specifically target the BCNGchannels. TABLE II Drugs and Toxins That Interact with Members of theVoltage-gated Ion Channel Family Therapeutic Compound Channel targetsSite of action Uses Local Anesthetics Na Pore (S6) Local (lidocaine,procaine, analgesia, etc.) arrhythmias diphenylhydantoin Na PoreSeizures Tetrodotoxin Na Pore Saxitoxin Na Pore α,β-Scorpion toxin NaActivation and Inactivation gates Dihydropyridines Ca (L-type) Porearrhythmias hypertensic angina Verapamil Ca (L-type) Pore Diltiazem Ca(L-type) Pore w-conotoxin Ca (N-type) Pore w-agatoxin Ca (P-type) PoreTetraethylammonium K Pore 4-aminopyridine K Pore charybdotoxin K Porehanatoxin K activation Gate amiodarone K ? arrhythmias 1-cis-diltiazemCNG Pore Rp-cAMPS CNG Binding site antagonist Sp-cAMPS CNG Binding siteagonist(Hille, 1992; Catterall, 1992; Roden, 1996)

From their amino acid sequence, these channels are likely to have threeimportant physiological properties that make them a priori attractivetargets for drug development. First, their gating should bevoltage-dependent and thus it should be sensitive to modulation of thevoltage-gating mechanism. Second, they possess a cyclic nucleotidebinding domain in their C-terminus and it is probable that their gatingwill be modulated by direct binding of cyclic nucleotides. This presentsa second target for drug targeting. Third, the unusual sequence of thepore forming domain of the BCNG channels should allow the ion conductionproperties of the channel to be selectively targeted.

If, the gating of the channels involves both the voltage-sensormachinery and the cyclic nucleotide binding site it is likely thatcoordinated drug regimes such that two compounds with low efficacy andeven low selectivity can combine to selectively target the BCNGchannels. Thus one compound that alone would have weak pharmacologicaleffects on many voltage-activated channels combined with one that has asimilarly weak effect on the various cyclic nucleotide binding pocketscould be applied together. As no class of molecules is currently knownthat functionally combines BOTH of these structural elements—with theanticipated exception of the BCNG channels—it is likely that such aregime would lead to a highly efficacious and selective targeting ofchannels containing the BCNG subunits. Selective intervention againstBCNG sub-types should also be possible.

The regulation of these channels through drugs provides a uniqueopportunity for regulating electrical activity associated with diseasesas diverse as epilepsy and cardiac arrhythmias. Moreover, the cyclicnucleotide binding domain of these channels provides a uniquepharmacological target that could be used to develop novel, specific,cyclic nucleotide agonists or antagonists to upregulate or downregulatechannel function

Drugs can modulate voltage-dependent gating—coupled with CNG to achieveselectivity.

Cell lines expressing BCNG-1, mBCNG-2, mBCNG-3, mBCNG-4, hBCNG-1 andhBCNG-2 offer the promise of rapid screening for compounds that interactwith the channels. To identify drugs that interact with the cyclicnucleotide binding domain, this region could be expressed selectively inbacteria and then purified. The purified protein fragment could then beused in standard ligand-binding assays to detect cyclic nucleotideanalogs that bind with high affinity.

Functional effects of drugs on channel opening or ion permeation throughthe pore are tested using whole cell patch clamp of mammalian cell linesexpressing the various BCNG genes. Where the BCNG channels resemble theCNG channels, they exhibit significant permeability to calcium. Thispermits a high throughput screen in which channel function is assessedby imaging intracellular calcium concentration. Drugs that increasechannel opening also increase internal calcium.

Example 4 Expression of Functional BCNG Proteins

As the BCNG genes appear to encode an ion channel, mBCNG-1 is expressedin Xenopus oocytes by injection of cRNA and current is recorded usingboth two electrode voltage clamp and excised inside-out patch clampapproaches. A lack of current may result from a failure of correcttrafficking or proper post-translational modification. In an attempt toover come such problems and to obtain functional expression of the BCNGchannels, alternative expression systems are utilized. BCNG channels areexpressed in a human cell line such as HEK293 cells, or in an insectcell line such as SF9 cells. Proper post-translational modificationprovided by the mammalian HEK293 cells or the efficient proteinproduction of the insect cell system, may be required to yield afunctional ion channel. As each new subunit of the BCNG family is clonedthey are expressed in each of the systems described above. Some membersof the family may form functional homomultimers (eg. as is seen withsome subunits of cyclic nucleotide gated channels and the some of theneuronal nicotinic acetylcholine receptor subunits).

Cloning Additional Subunits that Interact with the BCNG Proteins to GiveRise to Functional Current

Coexpression of BCNG channel subunits and candidate proteins. Althoughthe BCNG clones show considerable homology, it is possible that thesubunits that are expressed in the same tissue form heteromultimericproteins with each other and the homomultimers are non-functional. Inorder to delineate the functional roles of new BCNG subunits, new BCNGsubunits are coexpressed with BCNG subunits that have overlapping tissuedistribution. Voltage-gated K⁺ channels and cyclic nucleotide-gatedchannels both form heteromultimers. In some cases, the subunits can formcomplexes with completely distinct proteins (eg. KvLQT1 withMinK—(Barhanin, et al., 1996; HERG with MinK McDonald, et al., 1997);IrK6.1 and IrK6.2 with the sulphonylurea receptor—Isomoto, et al., 1996;Inagaki, et al., 1996). BCNG proteins may assemble with subunits such asMinK or ERG like subunits. Candidate proteins are selected on the basisof overlapping tissue distribution and likelihood based on knownfunctional properties. For example, Kv1.2 shows overlapping distributionwith mBCNG-1 even at the subcellular level (Sheng, et al., 1994; Wang,et al., 1994).

Coexpression with PolyA+ mRNA. If another protein is required to form afunctional heteromultimer with the BCNG channel proteins, co-expressionwith size fractionated mRNA from tissue (eg. heart, brain, muscle orkidney) where the appropriate BCNG subunit is expressed (as shown byNorthern blot analysis) should result in a unique current inelectrophysiological currents when the BCNG RNA is coinjected with themRNA from the tissue.

Alternative strategies to clone subunits that will permit functionalexpression of the BCNG channels include low stringency homologyscreening of the appropriate libraries using BNA nucleotide probesderived from the current BCNG genes or PCR amplification from genomic orcDNA using degenerate oligonucleotides based on the known BCNG genes.

Yet another method to isolate other channel subunit proteins that maycoassemble with identified BCNG family members is to use the yeast twohybrid system (Fields and Soug, 1989). This system was initially used toclone mBCNG-1 based on its interaction with the n-src SH3 domain.Conserved cytoplasmic N- and C-terminal domains from BCNG channelproteins are used as the ‘bait’ in the yeast two hybrid system. N- andC-terminal fragments will be subcloned in the plasmid pEG202 (Zervos etal., 1983).

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1. An isolated nucleic acid molecule having sequence encoding a BCNG ionchannel protein or a portion thereof.
 2. The nucleic acid molecule ofclaim 1, wherein the BCNG ion channel protein is mBCNG-1, mBCNG-2,mBCNG-3, mBCNG-4, hBCNG-1 or hBCNG-2.
 3. The nucleic acid molecule ofclaim 1, wherein the nucleic acid is DNA or RNA.
 4. The nucleic acidmolecule of claim 3, wherein the DNA is cDNA.
 5. The nucleic acidmolecule of claim 4, wherein the cDNA has substantially the same codingsequence as the coding sequence shown in SEQ. ID. No.: 1, SEQ. ID. No.:3, SEQ. ID. No.: 5, SEQ. ID. No.: 7, SEQ. ID. No.: 9, or SEQ. ID.No.:11.
 6. A vector comprising the nucleic acid molecule of claim
 4. 7.The vector of claim 6, wherein the vector is a virus or a plasmid.
 8. Ahost vector system for the production of a mammalian BCNG molecule whichcomprises the vector of claim 6 in a suitable host.
 9. The host vectorsystem of claim 8, wherein the suitable host is a bacterial cell, aeukaryotic cell, a mammalian cell or an insect cell.
 10. A nucleic acidmolecule capable of specifically hybridizing with the nucleic acidmolecule of claim
 1. 11. An isolated BCNG protein.
 12. The protein ofclaim 11, wherein the BCNG protein is mBCNG-1, mBCNG-2, mBCNG-3,mBCNG-4, hBCNG-1 or hBCNG-2.
 13. The protein of claim 11, wherein theBCNG protein has substantially the same amino acid sequence as the aminoacid sequence shown in SEQUENCE ID NO.: 2, SEQ. ID. No.: 4, SEQ. ID.No.: 6, SEQ. ID. No.: 8, SEQ. ID. No.: 10, or SEQ. ID. No.:
 12. 14. Acomposition comprising a nucleic acid, having sequence encoding a BCNGprotein or a portion thereof and a carrier.
 15. The composition of claim14, wherein the nucleic acid comprises substantially the same codingsequence as the coding sequence shown in SEQ. ID. No.: 1, SEQ. ID. No.:3, SEQ. ID. No.: 5, SEQ. ID. No.: 7, SEQ. ID. No.: 9, SEQ. ID. No.:11 orportion thereof.
 16. A composition comprising a BCNG protein or portionthereof and a carrier.
 17. The composition of claim 16, wherein the BCNGprotein comprises substantially the same amino acid sequence as theamino acid sequence shown in SEQ. ID. No.: 2, SEQ. ID. No.: 4, SEQ. ID.No.: 6, SEQ. ID. No.: 8, SEQ. ID. No.: 10, SEQ. ID. No.:12 or portionthereof.
 18. A method of identifying a nucleic acid encoding an ionchannel subunit-related protein in a sample which comprises detecting inthe sample a nucleic acid molecule encoding a BCNG-related protein,positive detection indicating the presence of an ion channel subunitrelated protein encoding nucleic acid molecule, thereby identifying anucleic acid encoding an ion channel subunit-related protein in thesample.
 19. The method of claim 18, wherein the nucleic acid moleculeencoding the ion channel subunit related protein is detected by sizefractionation.
 20. The method of claim 19, wherein the sizefractionation is effected by a polyacrylamide or an agarose gel.
 21. Themethod of claim 20, wherein the detection of the nucleic acid moleculeencoding the BCNG-related protein comprises contacting the sample with asecond nucleic acid molecule capable of hybridizing with the nucleicacid molecule encoding the BCNG-related protein, wherein the secondnucleic acid molecule is labeled with a detectable marker underconditions permitting the second nucleic acid molecule to hybridize withthe nucleic acid molecule encoding the BCNG-related protein, therebyidentifying the nucleic acid molecule in the sample.
 22. The method ofclaim 21, wherein the second nucleic acid molecule is capable ofhybridizing to mBCNG-1, mBCNG-2, mBCNG-3, mBCNG-4, hBCNG-1 or hBCNG-2.23. The method of claim 21, wherein the detectable marker is aradiolabeled molecule, a fluorescent molecule, an enzyme, a ligand, or amagnetic bead.
 24. The method of claim 18, wherein the detection of thenucleic acid molecule encoding the BCNG-related protein comprisesamplifying the nucleic acid molecule encoding the BCNG-related protein,thereby identifying the nucleic acid molecule encoding the BCNG-relatedprotein in the sample.
 25. The method of claim 24, wherein theamplification of the nucleic acid molecule encoding the BCNG-relatedprotein comprises contacting the sample with at least one primer capableof hybridizing to mBCNG-1, mBCNG-2, mBCNG-3, mBCNG-4, hBCNG-1 or hBCNG-2under conditions suitable for polymerase chain reaction.
 26. The methodof claim 25, wherein the amplified nucleic acid molecule encoding theBCNG-related protein is detected by size fractionation.
 27. The methodof claim 26, wherein the size fractionation is via a polyacrylamide gelor an agarose gel.
 28. A method for evaluating the ability of a compoundto modulate an ion channel associated with a condition in a subjectwhich comprises: (a) contacting a cell which expresses a BCNG-relatedprotein in a cell culture with a compound; (b) determining the amount ofBCNG-related protein expression in the cell culture; and (c) comparingthe amount of BCNG-related protein expression determined in step (b)with the amount determined in the absence of the compound so as toevaluate the ability of the compound to modulate the ion channelassociated with the condition in the subject.
 29. The method of claim28, wherein the condition is a neurological condition, cardiovascularcondition, renal condition, pulmonary condition, or hepatic condition.30. The method of claim 28, wherein the condition is epilepsy,Alzheimer's Disease, Parkinson's Disease, long QT syndrome, sick sinussyndrome, age-related memory loss, cystic fibrosis, sudden deathsyndrome or a pacemaker rhythm dysfunction.
 31. The method of claim 28,wherein the BCNG-related protein comprises mBCNG-1, mBCNG-2, mBCNG-3,MBCNG-4, hBCNG-1 or hBCNG-2 or a portion thereof.
 32. The method ofclaim 28, wherein the cell is a cardiac cell, a kidney cell, a hepaticcell, an airway epithelial cell, a muscle cell, a neuronal cell, a glialcell, a microglial cell, an endothelial cell, a mononuclear cell, atumor cell, a mammalian cell, an insect cell, or a Xenopus oocyte. 33.The method of claim 28, wherein the compound is a peptide, apeptidomimetic, a nucleic acid, a polymer, or a small molecule.
 34. Themethod of claim 28, wherein the compound is bound to a solid support.35. A method for evaluating the ability of a compound to interact with aBCNG-related ion channel subunit protein which comprises: (a) contactinga cell which expresses a BCNG-related protein in a cell culture with acompound under conditions permissive to formation of a complex betweenthe compound and the BCNG-related protein; (b) determining the amount ofcomplex formed between the compound and the BCNG-related protein in thecell culture; and (c) comparing the amount of complex formed in step (b)with the amount formed in the absence of the compound so as to evaluatethe ability of the compound to interact with a BCNG-related ion channelsubunit protein.
 36. The method of claim 35, wherein the BCNG-relatedprotein comprises mBCNG-1, mBCNG-2, mBCNG-3, mBCNG-4, hBCNG-1 or hBCNG-2or a portion thereof.
 37. The method of claim 35, wherein the cell is acardiac cell, a kidney cell, a hepatic cell, an airway epithelial cell,a muscle cell, a neuronal cell, a glial cell, a microglial cell, anendothelial cell, a mononuclear cell, a tumor cell, a mammalian cell, aninsect cell, or a Xenopus oocyte.
 38. The method of claim 35, whereinthe compound is a peptide, a peptidomimetic, a nucleic acid, a polymer,or a small molecule.
 39. The method of claim 35, wherein the compound isbound to a solid support.
 40. A method for identifying whether acompound is capable of modulating a dysfunction in an animal comprising:(a) administering the compound to the animal under conditions permissiveto formation of a complex between the compound and the BCNG-relatedprotein; (b) determining the amount of complex formed between thecompound and the BCNG-related protein in the animal; and (c) comparingthe amount of complex formed in step (b) with the amount of complexformed in the animal in the absence of the compound so as to identifywhether the compound is capable of modulating the dysfunction in theanimal, a change in the amount of complex formed in the presence of thecompound indicating that the compound is capable of modulatingdysfunction in the animal.
 41. The method of claim 40, wherein thecondition is a neurological condition, cardiovascular condition, renalcondition, pulmonary condition, or hepatic condition.
 42. The method ofclaim 41, wherein the condition is epilepsy, Alzheimer's Disease,Parkinson's Disease, long QT syndrome, sick sinus syndrome, age-relatedmemory loss, cystic fibrosis, sudden death syndrome or a pacemakerrhythm dysfunction.
 43. A method of identifying a compound whichmodulates the activity of BCNG-related protein which comprises: (a)introducing the nucleic acid of claim 1 into an expression system andcausing the expression system to express the nucleic acid underconditions whereby an ion channel subunit protein is produced; (b)contacting the channel subunit protein with the compound; (c)determining the activity of the ion channel subunit protein; and (d)comparing the activity determined in step (c) with the activitydetermined in the absence of the compound, an increase or decrease inactivity in the presence of the compound indicating identification of acompound which modulates the activity of BCNG-related protein.
 44. Themethod of claim 43, wherein the activity determination step (c)comprises measuring the channel electrical current or intracellularcalcium level in the presence of the compound.
 45. The method of claim43, wherein the expression system comprises a cultured host cell. 46.The method of claim 45, wherein the cultured host cell is a cardiaccell, a kidney cell, a hepatic cell, an airway epithelial cell, a musclecell, a neuronal cell, a glial cell, a microglial cell, an endothelialcell, a mononuclear cell, a tumor cell, a mammalian cell, an insectcell, or a Xenopus oocyte.
 47. A method of treating a condition in asubject which comprises administering to the subject an effective amountof a compound which modulates the activity of BCNG-related proteinidentified by the method of claim 43, so as to treat the condition. 48.The method of claim 47, wherein the condition is a neurologicalcondition, cardiovascular condition, renal condition, pulmonarycondition, or hepatic condition.
 49. The method of claim 48, wherein thecondition is epilepsy, Alzheimer's Disease, Parkinson's Disease, long QTsyndrome, sick sinus syndrome, age-related memory loss, cystic fibrosis,sudden death syndrome or a pacemaker rhythm dysfunction.
 50. Apharmaceutical composition which comprises a compound capable ofmodulating BCNG-related protein activity identified by the method ofclaim 43, and a pharmaceutically acceptable carrier.
 51. Thepharmaceutical composition of claim 50, wherein the carrier is adiluent, an aerosol, a topical carrier, an aqueous solution, anonaqueous solution or a solid carrier.
 52. A method for treating acondition in a subject which comprises administering to the subject anamount of the pharmaceutical composition of claim 51, effective to treatthe condition in the subject.
 53. The method of claim 52, wherein thecondition is a neurological condition, renal condition, pulmonarycondition, hepatic condition, or cardiovascular condition.
 54. Themethod of claim 53, wherein the condition is epilepsy, Alzheimer'sDisease, Parkinson's Disease, long QT syndrome, sick sinus syndrome,age-related memory loss, cystic fibrosis, sudden death syndrome or apacemaker rhythm dysfunction.
 55. The method of claim 52, wherein thesubject is a human.
 56. An antibody which binds specifically to theprotein of claim
 11. 57. A cell line producing the antibody of claim 56.58. A method of identifying the protein of claim 11 in a samplecomprising: (a) contacting the sample with the antibody of claim 56under conditions permissive to the formation of a complex between theantibody and the protein; (b) determining the amount of complex formed;and (c) comparing the amount of complex formed in step (b) with theamount of complex formed in the absence of the antibody, the presence ofan increased amount of complex formed in the presence of the antibodyindicating identification of the protein in the sample.